Therapeutic targeting of phosphate dysregulation in cancer via the xpr1:kidins220 protein complex

ABSTRACT

The subject matter disclosed herein is generally directed to inhibition of XPR1 :KIDINS220-mediated phosphate export to treat cancer, in particular, ovarian and uterine cancers. The subject matter disclosed herein is also generally directed to determining cancer dependency on phosphate export by detecting the expression of SLC34A2. Compositions for inhibiting XPR1 :KIDINS220-mediated phosphate export are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/062,890, filed Aug. 7, 2020. The entire contents of theabove-identified application are hereby fully incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.CA242457 and CA212229 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD-5160WP_ST25.txt”;Size is 18,185 bytes and it was created on Aug. 6, 2021, is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The subject matter disclosed herein is generally directed to inhibitionof XPR1:KIDINS220-mediated phosphate export in the treatment of cancerand determining cancer dependency on phosphate export by detecting theexpression of SLC34A2.

BACKGROUND

Ovarian and uterine cancers are among the deadliest cancers that affectwomen, and while platinum-based chemotherapies and recently approvedPARP inhibitors show efficacy for some patients ²⁻⁴, outcomes in thesecancers have not improved greatly in the past twenty years ^(5,6).Clearly, new therapeutic options are needed.

While organismal phosphate homeostasis is well understood, the cellularcoordination of phosphate import, storage, and efflux is poorlyunderstood. Phosphate uptake is highly regulated and involves members ofthe SLC34 and SLC20 gene families ⁷⁻⁹. In yeast and plants, specializedvacuoles and metabolites coordinate phosphate storage, but thesepathways are not elucidated in human biology ^(10,11). Phosphate effluxis achieved via the only annotated human exporter - the Xenotropic andPolytropic Receptor 1 (XPR1) ¹². XPR1 is involved in phosphatehomeostasis of various tissues ¹³⁻¹⁵, although the mechanisms of itsregulation and function are poorly understood ^(16,17) _(.)

Citation or identification of any document in this application is not anadmission that such a document is available as prior art to the presentinvention.

SUMMARY

In one aspect, the present invention provides for a method of treatingcancer in a subject in need thereof comprising administering to thesubject one or more therapeutic agents capable of inhibiting ofXPR1:KIDINS220-mediated phosphate export. In certain embodiments, thecancer is selected from the group consisting of ovarian cancer, uterinecancer, breast cancer, bile duct cancer, liver and lung cancer. Incertain embodiments, the cancer is characterized by higher expression ofSLC34A2 in tumor tissue as compared to expression in normal tissue. Incertain embodiments, the one or more therapeutic agents inhibit theexpression or activity of XPR1, inhibit the expression or activity ofKIDINS220, and/or disrupt XPR1/KIDINS220 interaction. In certainembodiments, the one or more therapeutic agents comprise a receptorbinding domain (RBD) protein derived from an enveloped virusglycoprotein and capable of interacting with the XPR1 membrane receptor.In certain embodiments, the RBD protein is a fusion protein, wherein thefusion protein comprises a domain capable of dimerization and/orstabilization of the protein. In certain embodiments, the RBD protein isfused to an Fc domain, glutathione S-transferase (GST), and/or serumalbumin. In certain embodiments, the RBD protein is derived fromxenotropic or polytropic murine leukemia retrovirus (X- and P-MLV) Env.In certain embodiments, the RBD fusion protein comprises the amino acidsequence:

MLVMEGSAFSKPLKDKINPWGPLIVMGILVRAGASVQRDSPHQIFNVTWRVTNLMTGQTANATSLLGTMTDTFPKLYFDLCDLVGDYWDDPEPDIGDGCRTPGGRRRTRLYDFYVCPGHTVPIGCGGPGEGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKDQGPCYDSSVSSGVQGATPGGRCNPLVLEFTDAGRKASWDAPKVWGLRLYRSTGADPVTRFSLTRQVLNVGPRVPIGSVDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 1).

In certainembodiments, the one or more therapeutic agents comprise avector encoding for the RBD protein. In certain embodiments, the one ormore therapeutic agents comprise an antibody specific for XPR1, anantibody specific for KIDINS220, or an antibody specific to theXPR1/KIDINS220 protein complex. In certain embodiments, the antibodytargets a Walker A/B motif of KIDINS220. In certain embodiments, the oneor more therapeutic agents comprise a degrader molecule. In certainembodiments, the degrader molecule is a LYTAC molecule, whereby a cellsurface protein is targeted. In certain embodiments, the one or moretherapeutic agents comprise a genetic modifying agent capable ofinhibiting the expression of XPR1 or KIDINS220. In certain embodiments,the genetic modifying agent comprises a CRISPR-Cas system, a RNAi, azinc finger nuclease, a TALE system, or a meganuclease. In certainembodiments, the CRISPR-Cas system is a CRISPR-Cas base editing system,a prime editor system, or a CAST system. In certain embodiments, themethod further comprises administering to the subject one or more agentscapable of inhibiting the expression or activity of FGF23, capable ofinhibiting the suppression of SLC34A2, or capable of modulating one ormore genes up or down-regulated in response to XPR1 inhibition. Incertain embodiments, one or more therapeutic agents capable ofinhibiting XPR1:KIDINS220-mediated phosphate export are co-administeredwithin a standard of care treatment regimen. In certain embodiments, thestandard of care treatment regimen comprises surgery and chemotherapy.In certain embodiments, the standard of care treatment regimen comprisesadministration of an immunotherapy, checkpoint blockade therapy or aPARP inhibitor.

In another aspect, the present invention provides for a method oftreating cancer in a subject in need thereof comprising: detectingtumors sensitive to phosphate dysregulation by detecting increasedexpression of SLC34A2 relative to a control, wherein if the subject hasa tumor sensitive to phosphate dysregulation, including administrationof one or more therapeutic agents capable of inhibitingXPR1:KIDINS220-mediated phosphate export according to any embodimentherein; if the subject does not have a tumor sensitive to phosphatedysregulation, administering a standard of care treatment that does notinclude administration of one or more therapeutic agents capable ofinhibiting XPR1:KIDINS220-mediated phosphate export. In certainembodiments, the cancer is selected from the group consisting of ovariancancer, uterine cancer, breast cancer, bile duct cancer, liver and lungcancer. In certain embodiments, the standard of care treatment comprisesone or more of surgery, chemotherapy, immunotherapy, checkpoint blockadetherapy or administration of a PARP inhibitor. In certain embodiments,the method further comprises monitoring the efficacy of the treatmentcomprising detecting in a tumor sample obtained from the subject theexpression of one or more genes selected from the group consisting ofSLC34A2, SLC20A1 and FGF23, wherein the treatment is effective ifSLC34A2 and/or SLC20A1 are decreased, and/or FGF23 is increased. Incertain embodiments, the method further comprises monitoring theefficacy of the treatment comprising detecting increased morphologicalchanges associated with phosphate dysregulation in tumor cells obtainedfrom the subject, wherein the treatment is effective if increasedmorphological changes associated with phosphate dysregulation aredetected. In certain embodiments, the morphological changes associatedwith phosphate dysregulation comprise vacuole-like structures in tumorcells.

In another aspect, the present invention provides for a method ofdetermining whether a subject suffering from cancer has a tumorsensitive to phosphate dysregulation comprising detecting the expressionof SLC34A2 in a tumor sample obtained from the subject, wherein ifSLC34A2 expression is higher in the tumor sample as compared toexpression in normal tissue the tumor is sensitive. In certainembodiments, the method further comprises detecting PAX8. In certainembodiments, the method further comprises detecting any gene thatco-varies with SLC34A2. In certain embodiments, the cancer is selectedfrom the group consisting of ovarian cancer, uterine cancer, breastcancer, bile duct cancer, liver and lung cancer.

In another aspect, the present invention provides for a method ofdetermining whether a subject suffering from cancer has a tumorsensitive to phosphate dysregulation comprising detecting theamplification in XPR1 copy number in a tumor sample obtained from thesubject, wherein if XPR1 copy number amplification is detected in thetumor sample the tumor is sensitive. In certain embodiments, copy numberis detected by inference from a target sequencing panel at the XPR1locus on chromosome 1. In certain embodiments, the cancer is selectedfrom the group consisting of ovarian cancer, uterine cancer, breastcancer, bile duct cancer, liver and lung cancer.

In certain embodiments, detecting comprises one or more ofimmunohistochemistry (IHC), in situ RNA-seq, quantitative PCR, RNA-seq,CITE-seq, western blot, Fluorescence In Situ Hybridization (FISH),RNA-FISH, mass spectrometry, or FACS.

In another aspect, the present invention provides for a method foridentifying an agent capable of inhibiting XPR1:KIDINS220-mediatedphosphate export, comprising: applying a candidate agent to a cancercell or cell population; and detecting modulation of phosphate efflux inthe cell or cell population by the candidate agent, thereby identifyingthe agent.

In another aspect, the present invention provides for a method foridentifying a cancer sensitive to inhibition of XPR1:KIDINS220-mediatedphosphate export, comprising: applying an inhibitor ofXPR1:KIDINS220-mediated phosphate export to a cancer cell or cellpopulation; and detecting the phosphate concentration in the cell orcell population, wherein the cancer is sensitive if the phosphateconcentration is increased as compared to a control cell or populationnot treated with the inhibitor. In certain embodiments, the inhibitor ofXPR1:KIDINS220-mediated phosphate export is one or more therapeuticagents according to any embodiment herein. In certain embodiments, thecancer cell or population is obtained or derived from a subject in needthereof.

These and other aspects, objects, features, and advantages of theexample embodiments will become apparent to those having ordinary skillin the art upon consideration of the following detailed description ofexample embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

An understanding of the features and advantages of the present inventionwill be obtained by reference to the following detailed description thatsets forth illustrative embodiments, in which the principles of theinvention may be utilized, and the accompanying drawings of which:

FIGS. 1 - XPR1 is a dependency in ovarian cancer in the context ofSLC34A2 overexpression. a. Across 733 cancer cell lines (DepMap Avana20Q1), the predictability and selectivity of cell line killing acrossall ~17,000 genes tested. Teal dots represent dependencies in ovariancancer cell lines. The inset shows the dependency profile for XPRR1. b.XPR1 and SLC34A2 expression and dependency profiles across all celllines, approximately ranked by decreasing dependency on XPR1. c. Becauseof their relative directionalities of phosphate transport, Applicantshypothesize that XPR1 inactivation is toxic because of intracellularphosphate accumulation in SLC34A2-high ovarian cancer. d. Viabilityeffects after inactivation of XPR1, scaled compared to negative andpositive control genes. e. Genome-scale CRISPR/Cas9 screen combined withiInactivation of XPR1 is combined — pairwise — with a genome-scale lossof the function screen to find potential ‘modifier’ genes for thedependency. Beta scores (determined by MaGeCK MLE) represent the changein representation for each gene from the initial library to the finaltimepoint. Teal points are genes significant only when combined withXPR1 inactivation, while mauve points are were significantly changed inthe XPR1 and in the control arms (such as the tumor suppressor CDKN2A).f. The SLC34A2 status of normally XPR1-resistant (ES2) or XPR1-sensitive(EMTOKA and OVISE) cell lines was modified by overexpression orknockout, and the XPR1 dependency was evaluated as in panel d.

FIGS. 2 - Validation of the XPR1 dependency in SLC34A2-overexpressingovarian cancer cell lines using shRNA. a. Bottom, a bubble plot showingthe expression of SLC34A2 across all cancer cell lines, with the degreeof dependency on XPR1 encoded by the size and color of the point (moredependent lines have larger points with deeper hues). Note that lungcancer cell lines do not display dependency on XPR1, despite highexpression of SLC34A2, and that some highly dependent cell lines do notexpress high levels of SLC34A2. Top, the Pearson correlation (R²)between SLC34A2 mRNA expression and XPR1 dependency within every lineagerepresented by at least 10 different cell lines. b. Three different celllines were engineered with doxycycline-inducible shRNA targeting XPR1(shXPR1_2 and shXPR1_4) or seed-matched control shRNA (shSeed_2 andshSeed_4) which have the same seed sequence but should not suppress XPR1mRNA. Three days after induction of shRNA, cellular lysates wereanalyzed by protein levels for KIDINS220 (see FIGS. 11 ), SLC34A2 (seeFIGS. 9 ), and XPR1. Protein levels normalized to vinculin are expressedbelow each band. c. Viability effect of suppression of XPR1 using shRNAreagents is highly penetrant. Cells were plated at low density andtreated with doxycycline to induce expression of shRNA. 14 days afterplating, surviving cells were stained with crystal violet. Note thatshXPR1 reagents effectively suppress growth, but shSeed reagents have noeffect on cellular growth. d. Seven days after induction of shRNA,viability was measured Quantification of using Cell Titer Glo (Promega).IGROV1 and OVISE both express high levels of SLC34A2 and are predictedto be dependent on XPR1; A2780 does not.

FIGS. 3 - A genome-scale CRISPR/Cas9 screen validates the relationshipbetween XPR1 dependency in the context of high expression of SLC34A2. a.Outline of the experimental method for a genome-scale, dual-knockoutmodifier screen. OVISE (without Cas9 expression) is engineered to stablyexpress sgRNA targeting XPR1. Upon introduction of “all-in-one”lentivirus, containing both Cas9 ORF and a second sgRNA, both genes aresimultaneously cut by Cas9. Applicants used three sgRNA: one targeting agene desert on chromosome 2 (sgChr2-2) and two targeting XPR1 (sgXPR1_1and sgXPR1_2) and infecting the cells with the Brunello genome-scalesgRNA library. 15 days after infection, Applicants collected cellpellets (far smaller in the sgXPR1 arms because of XPR1-dependent celldeath) and sequenced the sgRNA. b. Western confirmation of dual-knockoutof XPR1 and SLC34A2. The three cell lines used in the genome-scalescreen were infected with “all-in-one” lentivirus expressing control-,XPR1-, or SLC34A2-targeting sgRNA. Note that in the sgXPR1 “anchor” celllines, XPR1 is suppressed with the control virus, indicating that thefirst infection provides XPR1-targeting sgRNA and the second infectionprovides Cas9 protein. NIC stands for “no-infection control’. c.Arm-level results of the genome-scale modifier screen. See methods forfull analysis details. Beta-scores represent the extent to which a genewas enriched or depleted relative to all other genes.

FIGS. 4 - XPR1 and SLC34A2 are overexpressed in patient samples and XPR1dependency is retained in mouse xenograft studies. a. SLC34A2 mRNAexpression is compared in normal (GTEx), tumor (TCGA), and cancer cellline (CCLE) samples. For CCLE cell lines, the XPR1-dependency status(CERES < -0.5) is indicated. Note that Fallopian tube is the tissue oforigin for many ovarian/uterine cancers. b. XPR1 copy number heatmap fora ~2.5 Mb region of chromosome 1 indicating XPR1 amplification in serousovarian cancer (TCGA OV). c. As in panel a, XPR1 mRNA expression iscompared across the indicated tissues. For TCGA samples, the XPR1 copynumber status (GISTIC) is indicated. d. In a CRISPR/Cas9 competitiontumor formation assay in the ovarian cancer cell line OVISE, thedepletion of the XPR1 and the indicated genes is plotted. GPX4 was usedas a metabolic dependency gene. PAX8 is a benchmark dependency in manyovarian cancers. POLR2D is a pan-essential positive control. The grayboxes represent the variability in 15 different control sgRNA whichtarget non-gene sites in the human genome. e. Same as panel d, but withthe uterine cancer cell lines SNGM. f. The tumor growth of a model ofdisseminated ovarian carcinomatosis after XPR1 suppression usingdoxycycline-inducible shXPR1_2. The inset shows the full growth curveson a linear scale, while the larger image is the percent growth aftertreatment. g. Same as in f, but with the non-targeting shSeed_2.

FIGS. 5 - SLC34A2 and XPR1 overexpression in ovarian cancer are likelydriven by PAX8. a. Comparison of SLC34A2 expression across tissues.Using the combined GTEx, TCGA, and CCLE dataset as in FIGS. 4A and G,the differential expression of SLC34A2 in each tissue relative to allother tissues is compared. The relevant gynecological tissues (fallopiantube, ovary, and uterus) are highlighted in teal. b. PAX8 expression isincreased in tumor samples relative to normal tissues. In the same wayas in FIG. 4 a , PAX8 expression is compared across the indicated tissuesamples. q-values indicate the likelihood of the indicated populationshaving the same PAX8 expression according to a Wilcoxon ranked sums testwith Bonferroni correction for multiple comparisons. c. PAX8 and SLC34A2expression are highly correlated in fallopian tube, ovarian and uterinetissues. d. XPR1 copy number heatmap for a ~2.5 Mb region of chromosome1 indicating XPR1 amplification in TCGA Uterine Corpus EndometrialCarcinoma²⁸. Each patient sample is represented by a horizontal line.Red indicates copy gain and blue indicates copy loss. Dashed verticallines are the location of indicated genes. Data are a subset of thesamples with rank ordered by highest copy gain to indicate both focaland chromosome arm-level gains.

FIGS. 6 - The XPR1 dependency is not affected by phosphate levels in thetissue culture medium. a. The concentration of phosphate in the growthmedium of Dependency Map cell lines does not determine the extent ofXPR1 dependency. Concentrations of phosphate were estimated frommanufacturer formulations (see methods). b. Experimental procedure formanipulating tissue culture medium and assessing its effect on XPR1dependency. The same parental cancer cell line was engineered to expressfirefly luciferase and Cas9, or renilla luciferase alone. After one weekof growing the cell lines in low-phosphate RPMI 1640, the two variantswere mixed together and infected with sgRNA-encoding lentivirus. Afterselection for lentivirus-infected cell lines, the initial representationof Cas9:parental cells was determined by measuring the ratio ofFirefly:Renilla luciferase using a DualGlo assay (Promega). One weekafter infection (Day 16 of the protocol), the extent to which geneticperturbation was detrimental to cell viability was determined using theDualGlo. c. The XPR1 dependency is maintained in a low phosphate mediumcondition. SNGM (endometrial cell line dependent on XPR1) and ES2 (clearcell ovarian cell line without XPR1 dependency) were profiled in theassay outlined in panel b. Note that the CERES score -displayed belowthe plot - represents the viability defect of the cell line 21 daysafter knockout of XPR1 and growth in the indicated growth medium.

FIGS. 7 - In vivo CRISPR/Cas9 competition assays for target validationin mouse xenografts. a. Experimental design for in vivo competitionassays. Using a rapid infection and selection protocol, pooled sgRNA canbe introduced via lentivirus into cancer cell lines and inoculated assubcutaneous xenograft and the effect of gene knockout can be evaluatedin a more physiologically-relevant environment than tissue culture. b.After rapid infection with pooled sgRNA, 8 million SNGM cells wereinoculated as subcutaneous xenografts and allowed to grow. Tumor tissuewas harvested at the indicated time points. c. Same as in d, but for theexperiment using the OVISE cancer cell line. d. sgRNA abundance in tumorxenografts was evaluated by PCR and next-generation sequencing analysis,and the fold change compared to the early time point is shown as aheatmap for all of the negative control genes as well as any gene witha >4 fold change in abundance in any of the screens. Same as in d, butfor the experiment using the OVISE cancer cell line.

FIGS. 8 - XPR1 suppression halts growth in disseminated ovariancarcinomatosis. a. Experimental design to assess the effect of XPR1suppression on established intraperitoneal ovarian carcinomatosis.IGROV1 constitutively expressing luciferase were engineered to induciblyexpress shXPR1_2 (to suppress XPR1) or shSeed_2 (control). Because thein vivo growth kinetics for the model were unknown, 4 different celldensities were inoculated in the peritoneal cavity of mice and tumorgrowth was monitored using bioluminescent imaging. After three weeks oftumor growth, one mouse from each inoculation group was fed withdoxycycline chow to induce expression of shRNA. Animals developedascites within 2-3 weeks after treatment, and so the study wasterminated. b. The growth rate of IGROV1 cells engineered with shXPR1_2was not dependent on the number of cells inoculated. c. Same as in panelb, but with IGROV1 cells engineered with shSeed_2. d. At the time oftreatment, the tumor burden was equivalent across the four differentgroups. e. Number of animals per treatment and shRNA group which haddeveloped ascites. f. IGROV1 cells, constitutively expressingluciferase, are detected on the indicated organs after studytermination. Here, the cells were taken from a mouse from the shXPR1_2-Dox group.

FIGS. 9 — Transcriptional profiling highlights a phosphate homeostaticresponse to XPR1 inactivation in SLC34A2 overexpressing ovarian cancer.a. At various time-points after treatment with doxycycline and inductionof shRNA, the intracellular phosphate was measured in OVISE and IGROV1cell lines. b. UMAP projection of MixSeq scRNA-sequencing results tocompare multiplexed cancer cells after XPR1 inactivation. c. Middle, thelog-fold change of the top 500 differentially expressed genes afterregressing out the effect of cell cycle. Left, summary annotations foreach cell line include XPR1 dependency (XPR1 CERES), the overalltranscriptional change (Average LFC), and the degree of cell cyclearrest observed in the single cell data (ΔG0/G1). Right, the correlationbetween cell lines of the overall transcriptional change. d. Themeasured transcriptional change in the indicated genes are plotted forthe highly correlated cell lines (see panel c) on the left and the otherfive cell lines on the right. e. Quantitation of SLC34A2 protein levels(measured by Protein Simple) after XPR1 inactivation. f. Radioactivephosphate uptake was measured in the OVISE ovarian cancer cell lineafter inactivation of the indicated genes.

FIGS. 10 — Transcriptional profiling reveals a phosphate-relatedhomeostatic response after XPR1 suppression. a. The viability of cells(as measured by total protein content) was measured in parallel withtotal phosphate. The total intracellular phosphate reported in FIG. 9Arefers to total phosphate measured divided by cellular viability. b.Experimental workflow to determine the transcriptional profile of XPR1inactivation across many different cancer cell lines. Cell lines werepooled according to their doubling time and infected in small pools withcontrol or XPR1 sgRNA at a high multiplicity of infection (MOI) in orderto maximize penetrance of XPR1 inactivation. The day after infection,cells were selected with puromycin and then grown until the fourth dayafter infection. At this point, the cells were counted and pooled perperturbation and incubated with cell-surface labeling antibodiescontaining RNA “hash-tags.” Cells across perturbations were then pooledand subjected to the 10X genomics single cell RNA sequencing (scRNAseq)pipeline. The single nucleotide polymorphisms (SNP) profile for eachcell was used to assign it to a particular cell line, while the“hash-tag” barcode was used to determine which perturbation that cellhad received. c. The total number of cells per cell line. Differencesare likely due to outgrowth of individual cell lines within each pool ordifferences in sensitivity to the high MOI used. d. The total number ofunique transcripts measured for each cell, as measured by uniquemolecular identifiers (UMIs) which were included in the preparation ofthe library before amplification and next generation sequencing. e. Thenumber of cells collected per cell line was compared between the twoconditions (control and XPR1 inactivation) to determine if a viabilitydefect is observed in the ~4 day timeframe of the experiment. f. Aftercell cycle regression, genes which were increased in expression afterXPR1 inactivation within dependent cell lines (IGROV1, EFE184, OVISE,RMGI, OVMANA, and OVCAR4) were analyzed by gene set enrichment. g. Sameas in f, but for genes which were decreased after XPR1 inactivation. h.Four days after induction of shXPR1 2 (IGROV1) or shXPR1_4 (OVISE) usingdoxycycline, the amount of secreted FGF23 was measured in theconditioned medium using ELISA. Note that SLC34A2 suppression isobserved at this time point.

FIGS. 11 — The phosphate efflux capacity of XPR1 is required forviability in SLC34A2-overexpressing cancer cell lines. a. Across 737cancer cell screened (DepMap Avana 20Q1), the viability defects of XPR1or KIDINS220 inactivation is plotted. b. After expression of theindicated XPR1 open reading frames in HEK-293Ts, the interaction betweenthe V5-tagged ORF and KIDINS220 was evaluated usingco-immunoprecipitation. See FIG. 13A for the design of these constructs.c. After genetic inactivation of XPR1 or KIDINS220, the localization ofXPR1-V5 was determined using immunofluorescence. Note that sgXPR1_1inactivates both endogenous XPR1 and the XPR1-V5 ORF, and so no stainingshould be expected. d. Three days after genetic inactivation of XPR1 orKIDINS220, cells were loaded with radioactive phosphate, and then effluxwas measured for 30 minutes after. e. The indicated XPR1 constructs weretested for their ability to rescue knockout of endogenous XPR1. TheL218S mutation in XPR1 has previously been shown to only have ~50% theefflux capacity of XPR1. f. Phase contrast images of ‘vacuole-like’phenotype 4-5 days after XPR1 inactivation. Arrowheads indicate thelocation of ‘vacuole-like’ structures. Scale bars = 200 um. g. Theacidic dye Lysotracker was used to stain live cells five days afterinactivation of XPR1. h. Transmission electron micrographs of“vacuole-like” structures (labeled V) or lysosomes (Lys) in OVISE cancercells after XPR1 inactivation.

FIGS. 12 — XPR1 and KIDINS220 are co-expressed and co-precipitated inlarge-scale datasets. a. Validation of KIDINS220 dependency wasperformed in ovarian and uterine cancer cell lines with a range ofSLC34A2 expression as in FIG. 1D. b. SLC34A2 expression is necessary andsufficient for KIDINS220 dependency, performed as in FIG. 1F. c. XPR1and KIDINS220 mRNA levels are highly correlated across many tissues.Using GTEx, TCGA, and CCLE data, the Pearson correlation was calculated.d. XPR1 and KIDINS220 suppression both cause increased intracellularphosphate, which is dependent on SLC34A2 expression. Five days afterinfection with the indicated sgRNA, intracellular phosphate wasdetermined as in FIG. 9A. e. High throughput protein:protein interactiondatabases implicate XPR1 and KIDINS220 as part of a protein complex. Theinteracting partners of XPR1 and KIDINS220 were downloaded from theBioGrid and Bioplex databases and compared. Genes which were present inthe interactomes of XPR1 or KIDINS220 consistently across multipledatasets are called out as text.

FIGS. 13 - XPR1 domain mutants vary in localization and KIDINS220 a.XPR1 WT refers to the 696 amino acid protein produced by NM_004736 (theonly isoform detected), while XPR1 (short) refers to the 631 amino acidproduct of NM_001135669. All constructs have C-terminal V5 tags forimmunoprecipitation, western blotting, and immunofluorescent detection.b. Immunofluorescent localization of some of the XPR1 mutants testedusing the V5 epitope tag. Left, WT XPR1 localizes to the secretorypathways as well as puncta within the cytoplasm. Middle, XPR1 (short)staining appears to be far more diffuse. The L218S mutation has the samelocalization pattern as WT XPR1, consistent with proper trafficking,similar to what has been observed previously (see main text). c.Co-immunoprecipitation of XPR1 domain mutants with endogenous KIDINS220in 293s. This is an uncropped version of FIG. 9B, including the SPXdomain only constructs (lanes 6-7). Right, the whole cell extract (WCE)from the experiment, indicating differing expression levels ofKIDINS220. It should be noted that much of the background signal isattributed to immunoblotting for endogenous XPR1 in the same experiment.Green arrows indicate the expected molecular weight of the ORF, incontrast to degradation products. d. Western blot validation ofguide-resistant ORF. OVISE.Cas9 cells (parental, left, or overexpressingthe WT XPR1 ORF, right, used in FIG. 9E) were infected with the indictedsgRNA and harvested 5 days after infection. The XPR1 ORF includes amutation to block binding of sgXPR1_2 but not sgXPR1_1 (note thesuppression of both isoforms with sgXPR1_1 but only endogenous XPR1 withsgXPR1 _2).

FIGS. 14 - Common organellar stains do not label “vacuoles”. a. 6 daysafter infection with lentivirus encoding sgXPR1_2, OVISE.Cas9 andSNGM.Cas9 cell lines were stained and imaged using the indicated dyesand stains. Arrowheads indicate the location of vacuole structures byphase contrast (not pictured). Positive staining was only observed forthe lysosomal dye LAMP1. b. Phase contrast images of ‘vacuole-like’phenotype 4-5 days after XPR1 inactivation. Arrowheads indicate thelocation of ‘vacuole-like’ structures. Scale bars = 200 um. c. Live cellDIC and confocal immunofluorescence images of XPR1 dependent cell lineOVISE 5 days after CRISPR inactivation of XPR1 or KIDINS220 vs controlsgRNA (sgChr2-2). Acidic organelles were detected with Lysotracker (red)and DNA with DAPI (blue). Arrowheads indicate the location of‘vacuole-like’ structures. d. Transmission electron micrographs ofOVISE.Cas9 5 days after XPR1 inactivation. Scale bars are indicatedwithin the figure. Lysosome and “vacuole-like” structures are indicatedby “Lys” and “V” respectively. e. Same as in c, but with KIDINS220inactivation.

FIG. 15 - Exemplary design of RBD fusion protein (51.2 kDa). The proteinincludes 33 residue signal sequence, 207 residue RBD from NZB strain, 4residue linker (GSVD), and 227 residue Fc from mouse IgG1.

FIG. 16 - Taxonomy of other viral strains that can infect murine XPR1.

FIGS. 17A-17G - XRBD is a drug-like inhibitor of the XPR1 and KIDINS220protein complex. a) XRBD protein inhibits XPR1-dependent phosphateefflux. Phosphate efflux was measured by incubating IGROV1 ovariancancer cell lines with RPMI without phosphate supplemented with³²P-labeled phosphate, washing the cells extensively, and thenincubating the cells in standard RPMI (~10 mM phosphate). After 30minutes, the conditioned medium was collected, and the cells were washedand then lysed. Phosphate efflux was calculated as the percent of ³²Pmeasured in the conditioned medium relative to the total 32P measured inthe lysate and conditioned medium. Where indicated, the cells werepretreated for 72 hours with 250 ng/mL Doxycycline to induce XPR1suppression via shXPR1_2. Where indicated, the cells were treated withvarying doses of XRBD protein during the ³²P uptake and efflux portionsof the experiment. Where indicated, “no phosphate” RPMI was used duringthe efflux portion to prevent the cells from exporting any ³²P. b)Western blot confirmation of stable knockout of XPR1 or KIDINS220 in293T cells. 293T cells were transiently transfected with an ‘all-in-one’plasmid containing both Cas9 and sgRNA targeting XPR1 or KIDINS220.Clonal populations were isolated by limiting dilution and analyzed bywestern blot. Wildtype human XPR1 (hXPR1) was re-expressed in theknockout background as a control. c) XRBD flow cytometry analysis of the293T cells analyzed in b. The cells were incubated with 20 nM XRBD-mFcfor 30 minutes at 37c, followed by extensive washing and incubation withan antimouse secondary conjugated to AlexaFluor488 (Invitrogen). Left,histograms from at least 10,000 single cells. Right, the medianfluorescent intensity of the populations shown on left is displayed as aheatmap. d) XRBD treatment causes viability defects in ovarian cancercell lines. The indicated cell lines were treated with the XRBD proteinat the indicated concentrations and cellular viability was assessed fivedays later by Cell Titre Glo (CTG, Promega). Right, a heatmap comparingeach cell lines’ XPR1 inactivation sensitivity (assessed by CRISPRviability assays) and XRBD sensitivity (decrease in cellular viabilityafter five day treatment with the top dose of XRBD relative to vehiclecontrol). Below, the Pearson correlation coefficient between XPR1inactivation sensitivity and XRBD treatment. e) Western blot analysisand XRBD treatment in a small panel of lung cancer cell lines. 5 daysafter inactivation of XPR1 or SLC34A2 in the indicated cell lines, XPR1or SLC34A2 protein levels were analyzed by western blot. Below, cellularviability was assessed as in d. f) Glycerol gradient sedimentationanalysis of XPR1-contatiing native protein complexes with or withoutKIDINS220 inactivation. 293T cells, or the stable KIDINS220 knockoutcell lines profiled in b were lysed, and 1 mg of lysate was layered onto10-30% glycerol gradients and spun at 55,000 RPM in a SW55 rotor for 3.5hours at 4c to separate protein complexes by molecular weight. 24fractions were collected from each gradient in which low molecularweight complexes correspond to low fraction numbers (i.e., 1-7) andlarger protein complexes are at larger numbers. XPR1 was detected infractions by immunoblot. g) RT-PCR validation of FGF23. 2 or 4 daysafter induction of the indicated shRNA with doxycycline, RNA wasextracted from cells, reverse-transcribed into cDNA, and levels of XPR1,FGF23, or Vinculin (housekeeping control) were quantified bygene-specific primers. The data shown were normalized to Vinculin andare displayed as the Log2 fold-change relative to the matched timepointwithout doxycycline treatment (Ctl).

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS General Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure pertains. Definitions of common termsand techniques in molecular biology may be found in Molecular Cloning: ALaboratory Manual, 2^(nd) edition (1989) (Sambrook, Fritsch, andManiatis); Molecular Cloning: A Laboratory Manual, 4^(th) edition (2012)(Green and Sambrook); Current Protocols in Molecular Biology (1987)(F.M. Ausubel et al. eds.); the series Methods in Enzymology (AcademicPress, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D.Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988)(Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2^(nd) edition2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney,ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008(ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829);Robert A. Meyers (ed.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 9780471185710); Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March,Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed.,John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Janvan Deursen, Transgenic Mouse Methods and Protocols, 2^(nd) edition(2011).

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise.

The term “optional” or “optionally” means that the subsequent describedevent, circumstance or substituent may or may not occur, and that thedescription includes instances where the event or circumstance occursand instances where it does not.

The recitation of numerical ranges by endpoints includes all numbers andfractions subsumed within the respective ranges, as well as the recitedendpoints.

The terms “about” or “approximately” as used herein when referring to ameasurable value such as a parameter, an amount, a temporal duration,and the like, are meant to encompass variations of and from thespecified value, such as variations of +/-10% or less, +/-5% or less,+/-1% or less, and +/-0.1% or less of and from the specified value,insofar such variations are appropriate to perform in the disclosedinvention. It is to be understood that the value to which the modifier“about” or “approximately” refers is itself also specifically, andpreferably, disclosed.

As used herein, a “biological sample” may contain whole cells and/orlive cells and/or cell debris. The biological sample may contain (or bederived from) a “bodily fluid”. The present invention encompassesembodiments wherein the bodily fluid is selected from amniotic fluid,aqueous humour, vitreous humour, bile, blood serum, breast milk,cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph,perilymph, exudates, feces, female ejaculate, gastric acid, gastricjuice, lymph, mucus (including nasal drainage and phlegm), pericardialfluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skinoil), semen, sputum, synovial fluid, sweat, tears, urine, vaginalsecretion, vomit and mixtures of one or more thereof. Biological samplesinclude cell cultures, bodily fluids, cell cultures from bodily fluids.Bodily fluids may be obtained from a mammal organism, for example bypuncture, or other collecting or sampling procedures.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

Various embodiments are described hereinafter. It should be noted thatthe specific embodiments are not intended as an exhaustive descriptionor as a limitation to the broader aspects discussed herein. One aspectdescribed in conjunction with a particular embodiment is not necessarilylimited to that embodiment and can be practiced with any otherembodiment(s). Reference throughout this specification to “oneembodiment”, “an embodiment,” “an example embodiment,” means that aparticular feature, structure or characteristic described in connectionwith the embodiment is included in at least one embodiment of thepresent invention. Thus, appearances of the phrases “in one embodiment,”“in an embodiment,” or “an example embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment, but may. Furthermore, the particular features,structures or characteristics may be combined in any suitable manner, aswould be apparent to a person skilled in the art from this disclosure,in one or more embodiments. Furthermore, while some embodimentsdescribed herein include some but not other features included in otherembodiments, combinations of features of different embodiments are meantto be within the scope of the invention. For example, in the appendedclaims, any of the claimed embodiments can be used in any combination.

Reference is made to Bondeson, et al., “Phosphate dysregulation via theXPR1:KIDINS220 protein complex is a therapeutic vulnerability in ovariancancer” bioRxiv 2020.10.16.339374; doi.org/10.1101/2020.10.16.339374(posted to bioRxiv on Oct. 17, 2020). All publications, published patentdocuments, and patent applications cited herein are hereby incorporatedby reference to the same extent as though each individual publication,published patent document, or patent application was specifically andindividually indicated as being incorporated by reference.

OVERVIEW

Exploiting cancer-specific metabolic states is an attractive strategy tokill cancer cells while sparing normal tissues¹. Inorganic phosphate isa fundamental component of DNA, an intermediate metabolite in numerouspathways, and a key signaling molecule, yet perturbing phosphatehomeostasis has not been explored as a cancer therapeutic. By analyzingCRISPR/Cas9 loss of viability screens across many cancer cell lines,Applicants found that high expression of SLC34A2, a phosphate importer,renders cells sensitive to inactivation of the phosphate exporter XPR1.This surprising finding indicates that accumulation of inorganicphosphate may be toxic to cancer cells. Applicants extensively validatedthis synthetic lethal interaction in cancer cell lines and mousexenografts, and found evidence that many primary ovarian cancer tumorswould be sensitive to phosphate dysregulation. Mechanistically,Applicants also identified a transcriptional response aimed at restoringphosphate homeostasis. Applicants also identified an XPR1-bindingpartner (KIDINS220) required for phosphate efflux. Applicants alsoidentified novel vacuole structures which Applicants hypothesize areassociated with toxic accumulation of intracellular phosphate.Applicants also show that a viral XPR1 binding protein, receptor-bindingdomain (RBD), inhibits XPR1-dependent phosphate efflux and is adrug-like inhibitor of XPR1. Overall, these data illustrate novelmechanisms by which ovarian cancer cells maintain phosphate homeostasis,and that the perturbation of these mechanisms is an unappreciatedtherapeutic vulnerability in ovarian cancer.

Embodiments disclosed herein provide methods of treating and diagnosingcancers based on identification of the dependency onXPR1:KIDINS220-mediated phosphate export in tumor cells (e.g., tumorsthat overexpress SLC34A2). In an example embodiment, receptor-bindingdomain (RBD) inhibits XPR1-dependent phosphate efflux and is a drug-likeinhibitor of XPR1 that can be used to treat sensitive tumors. Sensitivetumors express increased SLC34A2, a phosphate importer, and do notsufficiently suppress phosphate import in response to XPR1/phosphateefflux inhibition to prevent cell growth inhibition. Applicants furtheridentified genes that co-vary (e.g., covariation) with SLC34A2 that canalso be used alone or in combination to detect cancers that arevulnerable to inhibition of XPR1:KIDINS220-mediated phosphate export(e.g., PAX8). As used herein, the term “co-vary” refers to genes thatare upregulated and downregulated together. As used herein “correlation”between genes refers to genes that co-vary. Applicants identified genesthat are differentially expressed in response to XPR1 inhibition. Forexample, SLC34A2 and SLC20A1 are suppressed and FGF23 is upregulated.Applicants determined that the tumor cells alter the expression in orderto restore phosphate homeostasis. Thus, targeting these mechanisms canfurther increase the vulnerability of sensitive tumors.

As used herein, XPR1 refers to xenotropic and polytropic retrovirusreceptor 1 (Also known as: IBGC6, SLC53A1, SYG1, X3). Exemplarysequences include the following NCBI accession numbers: NM_004736.4, NM001135669.2, NM 001328662.2, NP_004727.2, NP_001129141.1 andNP_001315591.1. XPR1 includes a SPX domain (N-terminal) and EXS domain(C-terminal) that can be targeted, in addition to targeting the entireprotein, by a therapeutic agent (e.g., small molecules, antibodies) oragent for detecting expression (e.g., antibodies). XPR1 is a phosphateexporter in metazoans, a function that does not require the SPX domain(see, e.g., Giovannini, et al., Inorganic phosphate export by theretrovirus receptor XPR1 in metazoans. Cell Rep. 2013;3(6):1866-1873;Legati, et al. Mutations in XPR1 cause primary familial braincalcification associated with altered phosphate export. Nat Genet.2015;47(6):579-581; Ansermet, et al., Renal Fanconi Syndrome andHypophosphatemic Rickets in the Absence of Xenotropic and PolytropicRetroviral Receptor in the Nephron . J Am Soc Nephrol.2017;28(4):1073-1078).

As used herein, KIDINS220 refers to kinase D interacting substrate 220(Also known as: ARMS, SINO). Exemplary sequences include the followingNCBI accession numbers: NM 020738.4, NM_001348729.2, NM_001348731.2,NM_001348732.2, NM 001348734.2, NM_001348735.2, NM 001348736.2, NM_001348738.2, NM_001348739.2, NM_001348740.2, NM_001348741.2, NM_001348742.2, NM_001348743.2, NM_001348745.2, NP_065789.1,NP_001335658.1, NP_001335660.1, NP_001335661.1, NP_001335663.1,NP_001335664.1, NP_001335665.1, NP_001335667.1, NP_001335668.1,NP_001335669.1, NP_001335670.1, NP_001335671.1, NP_001335672.1, andNP_001335674.1. KIDINS220 includes an Ankyrin repeat-containing domain(N-terminal) and KAP family P-loop domain that can be targeted by atherapeutic agent (e.g., small molecules, antibodies) or agent fordetecting expression (e.g., antibodies). The P-loop domain ischaracterized by two conserved motifs, termed the Walker A and B motifs.The Walker A motif, also known as the Walker loop, or P-loop, orphosphate-binding loop, is a motif in proteins that is associated withphosphate binding. The Walker B motif is a motif in most P-loop proteinssituated well downstream of the A-motif.

As used herein, SLC34A2 refers to solute carrier family 34 member 2(Also known as: NAPI-3B, NAPI-IIb, NPTIIb, PULAM). Exemplary sequencesinclude the following NCBI accession numbers: NM_006424.3, NM_001177998.2, NM_001177999.1, NP_006415.3, NP_001171469.2, andNP_001171470.1. Synthetic peptides derived from a complementaritydetermining region hypervariable domain amino acid sequence of ahumanized monoclonal antibody to NaPi2B transporter has been describedfor inhibiting tumor growth or treating cancer (US 9,193,797 B2).

As used herein, SLC20A1 refers to solute carrier family 20 member 1(Also known as: GLVR1, Glvr-1, PIT1, PiT-1). Exemplary sequences includethe following NCBI accession numbers: NM _005415.5 and NP_005406.3.

As used herein, PAX8 refers to paired box 8. Exemplary sequences includethe following NCBI accession numbers: NM_003466.4, NM_013952.4,NM_013953.4, NM_013992.4, NP_003457.1, NP_039246.1, NP_039247.1, andNP_054698.1.

As used herein, FGF23 refers to fibroblast growth factor 23 (Also knownas: ADHR, FGFN, HFTC2, HPDR2, HYPF, PHPTC). Exemplary sequences includethe following NCBI accession numbers: NM_020638.3 and NP_065689.1.

The present invention may be useful for the treatment of any cancerdependent on XPR1:KIDINS220-mediated phosphate export. In otherpreferred embodiments, the cancer has increased expression of SLC34A2 ascompared to normal tissue (e.g., ovarian cancer, uterine cancer, breastcancer, bile duct cancer, liver and lung cancer). In more preferredembodiments, the cancer is ovarian or uterine cancer. Detection ofSLC34A2 expression is further described in the diagnostic methodssection herein.

Exemplary cancers that may benefit from treatment with one or moreinhibitors of XPR1:KIDINS220-mediated phosphate export include, withoutlimitation, liquid tumors such as leukemia (e.g., acute leukemia, acutelymphocytic leukemia, acute myelocytic leukemia, acute myeloblasticleukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,acute monocytic leukemia, acute erythroleukemia, chronic leukemia,chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemiavera, lymphoma (e.g., Hodgkin’s disease, non-Hodgkin’s disease),Waldenstrom’s macroglobulinemia, heavy chain disease, or multiplemyeloma. The cancer may include, without limitation, solid tumors suchas sarcomas and carcinomas. Examples of solid tumors include, but arenot limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, cystadenocarcinoma, medullary carcinoma, epithelialcarcinoma, bronchogenic carcinoma, hepatoma, colorectal cancer (e.g.,colon cancer, rectal cancer), anal cancer, pancreatic cancer (e.g.,pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors),breast cancer (e.g., ductal carcinoma, lobular carcinoma, inflammatorybreast cancer, clear cell carcinoma, mucinous carcinoma), ovariancarcinoma (e.g., ovarian epithelial carcinoma or surfaceepithelial-stromal tumour including serous tumour, endometrioid tumorand mucinous cystadenocarcinoma, sex-cord-stromal tumor), prostatecancer, liver and bile duct carcinoma (e.g., hepatocelluar carcinoma,cholangiocarcinoma, hemangioma), choriocarcinoma, seminoma, embryonalcarcinoma, kidney cancer (e.g., renal cell carcinoma, clear cellcarcinoma, Wilm’s tumor, nephroblastoma), cervical cancer, uterinecancer (e.g., endometrial adenocarcinoma, uterine papillary serouscarcinoma, uterine clear-cell carcinoma, uterine sarcomas andleiomyosarcomas, mixed mullerian tumors), testicular cancer, germ celltumor, lung cancer (e.g., lung adenocarcinoma, squamous cell carcinoma,large cell carcinoma, bronchioloalveolar carcinoma, non-small-cellcarcinoma, small cell carcinoma, mesothelioma), bladder carcinoma,signet ring cell carcinoma, cancer of the head and neck (e.g., squamouscell carcinomas), esophageal carcinoma (e.g., esophagealadenocarcinoma), tumors of the brain (e.g., glioma, glioblastoma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma), neuroblastoma, retinoblastoma, neuroendocrine tumor,melanoma, cancer of the stomach (e.g., stomach adenocarcinoma,gastrointestinal stromal tumor), or carcinoids. Lymphoproliferativedisorders are also considered to be proliferative diseases. In certainexample embodiments, the cancer is ovarian cancer. In certain otherexample embodiments, the cancer is uterine cancer.

In another aspect, embodiments disclosed herein are directed to a methodof treating cancer based on determining if the subject has a tumorsensitive to phosphate dysregulation and administering one or moretherapeutic agents that target XPR1:KIDINS2200-mediated phosphate exportif the subject has a tumor sensitive to phosphate dysregulation. Inanother aspect, embodiments disclosed herein provide methods ofdetermining whether a subject has a tumor sensitive to phosphatedysregulation by detecting expression of SLC34A2 in a tumor sample ordetecting amplification in XPR1 copy number in a tumor sample.

THERAPEUTIC METHODS OF TARGETING CANCER DEPENDENCIES Therapeutic Agents

In certain embodiments, the present invention provides for one or moretherapeutic agents targeting XPR1:KIDINS220-mediated phosphate export.In preferred embodiments, the therapeutic agents are inhibitors ofXPR1:KIDINS220-mediated phosphate export. In certain embodiments, thetherapeutic agent blocks or disrupts the XPR1:KIDINS220 protein complexfrom functioning to export inorganic phosphate from a tumor cell. Incertain embodiments, the therapeutic agent blocks the XPR1:KIDINS220protein complex from forming. In certain embodiments, the therapeuticagent targets an extracellular domain of XPR1. In certain embodiments,the therapeutic agent targets the SPX domain or EXS domain of XPR1. Incertain embodiments, the therapeutic agent targets the Ankyrinrepeat-containing domain or KAP family P-loop domain (including theWalker A/B motifs) of KIDIN220.

In certain embodiments, targeting XPR1:KIDINS220-mediated phosphateexport may be made in combination with the current standard of care andmay provide for improved treatment and/or less toxicity. In certainembodiments, the one or more therapeutic agents comprise therapeuticpolypeptides, a small molecule inhibitor, small molecule degrader (e.g.,ATTEC, AUTAC, LYTAC, or PROTAC), antibody, antibody fragment,antibody-like protein scaffold, aptamer, genetic modifying agents (e.g.,CRISPR-Cas systems, TALENs, Zinc Finger Nucleases, Meganucleases), RNAior any combination thereof. In one example embodiment, the one or moretherapeutic agents comprise one or more therapeutic polypeptides. Inanother example embodiment, the therapeutic polypeptides are envelopreceptor-binding domains (RBD). In another example embodiment, the oneor more therapeutic agents comprises one or more antibodies specific toXPR1, specific to KIDINS220, or specific to the XPR1/KIDINS2200 complex.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound, orcombination of molecules or compounds, that confers some beneficialeffect upon administration to a subject. The beneficial effect includesenablement of diagnostic determinations; amelioration of a disease,symptom, disorder, or pathological condition; reducing or preventing theonset of a disease, symptom, disorder or condition; and generallycounteracting a disease, symptom, disorder or pathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested. As used herein “treating”includes ameliorating, curing, preventing it from becoming worse,slowing the rate of progression, or preventing the disorder fromre-occurring (i.e., to prevent a relapse).

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

For example, in methods for treating cancer in a subject, an effectiveamount of a combination of inhibitors targeting epigenetic genes is anyamount that provides an anti-cancer effect, such as reduces or preventsproliferation of a cancer cell or is cytotoxic towards a cancer cell. Incertain embodiments, the effective amount of an inhibitor targeting anepigenetic gene is reduced when an inhibitor is administeredconcomitantly or in combination with one or more additional inhibitorstargeting epigenetic genes as compared to the effective amount of theinhibitor when administered in the absence of one or more additionalinhibitors targeting epigenetic genes. In certain embodiments, theinhibitor targeting an epigenetic gene does not reduce or preventproliferation of a cancer cell when administered in the absence of oneor more additional inhibitors targeting epigenetic genes.

Therapeutic Polypeptides

In one example embodiment, a method of targeting cancer phosphatedependency comprises administering a therapeutic polypeptide. In certainembodiments, a polypeptide therapeutic is used to inhibit theXPR1:KIDINS220-mediated phosphate export. The therapeutic polypeptidemay inhibit XPR1:KIDINS220-mediated phosphate export by binding to XPR1,KIDINS220, or the XPR1/KIDINS200 complex. In one example embodiment, thetherapeutic polypeptide is an envelope-receptor-binding domain (“RBD”).In another example embodiment, the therapeutic polypeptide is anantibody or fragment or variant thereof.

Envelope-Receptor-Binding Domain (RBD)

In certain embodiments, the protein therapeutic is a receptor bindingprotein “RBD” protein that can bind to and inhibit the XPR1:KIDINS220protein complex. In certain example embodiments, the RBD protein isderived from an enveloped virus glycoprotein and capable of interactingwith the XPR1 membrane receptor. XPR1 is the xenotropic and polytropicreceptor for a variety of murine leukemia viruses (MLV or MuLV). Incertain embodiments, “RBD” is an about 238 residue fragment of theenvelope glycoprotein for a retrovirus, such as X-MLV, and fragmentinhibits XPR1 phosphate efflux (see, e.g., Giovannini, et al., Inorganicphosphate export by the retrovirus receptor XPR1 in metazoans. Cell Rep.2013;3(6):1866-1873). The RBD proteins can be modified as describedherein. In certain embodiments, the RBD can be a fusion protein. Forexample, the RBD can be an Fc fusion protein to increase stability invivo. In certain embodiments, the Fc domain promotes dimerization of theRBD fusion protein. In certain embodiments, the RBD protein can be afusion protein linked to glutathione S-transferase (GST), and/or serumalbumin (e.g., HSA or MSA). In certain embodiments, the GST domainpromotes dimerization of the RBD fusion protein (see, e.g., Tudyka andSkerra, Glutathione S-transferase Can Be Used as a C-terminal,Enzymatically Active Dimerization Module for a Recombinant ProteaseInhibitor, and Functionally Secreted Into the Periplasm of EscherichiaColi. Protein Science (1997), 6:2180-2187). Structural analyses haverevealed that glutathione S-transferase (GST) can form homodimers (Ji etal., Biochemistry. 1992 Oct 27; 31(42):10169-84; Reinemer et al., J MolBiol. 1992 Sep 5; 227(1):214-26; and Kaplan et al., Protein Sci. 1997Feb; 6(2):399-406).

FIG. 15 shows an exemplary RBD Fc fusion protein;

MLVMEGSAFSKPLKDKINPWGPLIVMGILVRAGASVQRDSPHQIFNVTWRVTNLMTGQTANATSLLGTMTDTFPKLYFDLCDLVGDYWDDPEPDIGDGCRTPGGRRRTRLYDFYVCPGHTVPIGCGGPGEGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKDQGPCYDSSVSSGVQGATPGGRCNPLVLEFTDAGRKASWDAPKVWGLRLYRSTGADPVTRFSLTRQVLNVGPRVPIGSVDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 2).

In certain embodiments, the mFc tag promotes dimerization of RBD. Incertain embodiments, the mFc tags promote recycling and stability of theRBD fusion protein. Giovannini, et al., 2013discloses KoRV, A-MLV, X-MLV(NZB-IU-6 strain), and PERV-A RBD C-terminally fused to a mouseimmunoglobulin (Ig) G1 Fc fragment and produced in HEK293T cells.Multiple MULVs use XPR1 with different species cross-reactivity: X-MLVsonly infect XPR1^(hum) P-MLVs use both XPR1^(mus) and XPR1^(hull). TheRBD protein can be derived from xenotropic or polytropic murine leukemiaretrovirus (X- and P-MLV) Env. FIG. 16 shows RBD proteins from otherviral strains that can infect through murine XPR1 and that areapplicable to the present invention. As used herein, an RBD protein thatis derived may be any non-natural hybrid of an RBD protein from anenveloped virus glycoprotein (e.g., non-natural hybrids X-and P-MLVs).As used herein “hybrid protein” refers to any protein that containssegments or parts from any two or more different proteins (e.g.,non-natural hybrids containing parts from both X- and P-MLVs), includinga non-naturally occurring modified protein.

Gene Therapy Embodiments For RBD

In one example embodiment, gene therapy may be used to express RBD intumor cells or the tumor microenvironment. In certain embodiments, genetherapy is used for subjects having tumors that overexpress SLC34A2. Asused herein, the terms “gene therapy,” “gene delivery,” “gene transfer”and “genetic modification” are used interchangeably and refer tomodifying or manipulating the expression of a gene to alter thebiological properties of living cells for therapeutic use.

In one example embodiment, a vector for use in gene therapy comprises asequence encoding an RBD protein and is used to deliver said sequence totumor cells. The vector may further comprise one or more regulatoryelements to control expression of the gene. The vector may furthercomprise regulatory/control elements, e.g., promoters, enhancers,introns, polyadenylation signals, Kozak consensus sequences, or internalribosome entry sites (IRES). The vector may further comprise a targetingmoiety that directs the vector specifically to tumor cells or the tumormicroenvironment. In another example embodiment, the vector may comprisea viral vector with a tropism specific for tumors.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. There are no limitations regarding the type of vectorthat can be used. The vector can be a cloning vector, suitable forpropagation and for obtaining polynucleotides, gene constructs orexpression vectors incorporated to several heterologous organisms.Suitable vectors include eukaryotic expression vectors based on viralvectors (e.g. adenoviruses, adeno- associated viruses as well asretroviruses and lentiviruses), as well as non-viral vectors such asplasmids.

In one example embodiment, the vector is a viral vector, whereinvirally-derived DNA or RNA sequences are present in the vector forpackaging into a virus (e.g., retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadenoassociated viruses). Viral vectors also include polynucleotidescarried by a virus for transfection into a host cell. Certain vectorsare capable of autonomous replication in a host cell into which they areintroduced (e.g., episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operably linked.Such vectors are referred to herein as “expression vectors.” Vectors forand that result in expression in a eukaryotic cell can be referred toherein as “eukaryotic expression vectors.” In another exampleembodiment, the vector integrates the gene into the cell genome or ismaintained episomally.

In one example embodiment, the vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques.

In one example embodiment, the vector is an mRNA vector (see, e.g.,Sahin, U, Kariko, K and Tureci, O (2014). mRNA-based therapeutics –developing a new class of drugs. Nat Rev Drug Discov 13: 759-780;Weissman D, Karikó K. mRNA: Fulfilling the Promise of Gene Therapy. MolTher. 2015;23(9):1416-1417. doi:10.1038/mt.2015.138; Kowalski PS, RudraA, Miao L, Anderson DG. Delivering the Messenger: Advances inTechnologies for Therapeutic mRNA Delivery. Mol Ther.2019;27(4):710-728. doi:10.1016/j.ymthe.2019.02.012; Magadum A, Kaur K,Zangi L. mRNA-Based Protein Replacement Therapy for the Heart. Mol Ther.2019;27(4):785-793. doi:10.1016/j.ymthe.2018.11.018; Reichmuth AM,Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine deliveryusing lipid nanoparticles Ther Deliv. 2016;7(5):319-334.doi:10.4155/tde-2016-0006; and Khalil AS, Yu X, Umhoefer JM, et al.Single-dose mRNA therapy via biomaterial-mediated sequestration ofoverexpressed proteins. Sci Adv. 2020;6(27):eaba2422). In an exemplaryembodiment, mRNA encoding for an RBD protein is delivered using lipidnanoparticles (see, e.g., Reichmuth, et al., 2016) and administereddirectly to a tumor. In an exemplary embodiment, mRNA encoding for anRBD protein is delivered using biomaterial-mediated sequestration (see,e.g., Khalil, et al., 2020) and administered directly to tumor tissue.Sequences present in mRNA molecules, as described further herein, areapplicable to mRNA vectors (e.g., Kozak consensus sequence, miRNA targetsites and WPRE).

Regulatory Elements

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operably-linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory element(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “operably linked” as usedherein also refers to the functional relationship and position of apromoter sequence relative to a polynucleotide of interest (e.g., apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of that sequence). Typically, an operablylinked promoter is contiguous with the sequence of interest. However,enhancers need not be contiguous with the sequence of interest tocontrol its expression. The term “promoter”, as used herein, refers to anucleic acid fragment that functions to control the transcription of oneor more polynucleotides, located upstream of the polynucleotidesequence(s), and which is structurally identified by the presence of abinding site for DNA-dependent RNA polymerase, transcription initiationsites, and any other DNA sequences including, but not limited to,transcription factor binding sites, repressor, and activator proteinbinding sites, and any other sequences of nucleotides known in the artto act directly or indirectly to regulate the amount of transcriptionfrom the promoter. A “tissue-specific” promoter is only active inspecific types of differentiated cells or tissues.

In another embodiment, the vector of the invention further comprisesexpression control sequences including, but not limited to, appropriatetranscription sequences (i.e., initiation, termination, promoter, andenhancer), efficient RNA processing signals (e.g., splicing andpolyadenylation (polyA) signals), sequences that stabilize cytoplasmicmRNA, sequences that enhance translation efficiency (i.e. Kozakconsensus sequence), and sequences that enhance protein stability. Agreat number of expression control sequences, including promoters whichare native, constitutive, inducible, or tissue-specific are known in theart and may be utilized according to the present invention.

In another embodiment, the vector of the invention further comprises apost-transcriptional regulatory region. In a preferred embodiment, thepost-transcriptional regulatory region is the Woodchuck Hepatitis Viruspost-transcriptional region (WPRE) or functional variants and fragmentsthereof and the PPT-CTS or functional variants and fragments thereof(see, e.g., Zufferey R, et al., J. Virol. 1999; 73:2886-2892; and KappesJ, et al., WO 2001/044481). In a particular embodiment, thepost-transcriptional regulatory region is WPRE. The term “Woodchuckhepatitis virus posttranscriptional regulatory element” or “WPRE”, asused herein, refers to a DNA sequence that, when transcribed, creates atertiary structure capable of enhancing the expression of a gene (see,e.g., Lee Y, et ah, Exp. Physiol. 2005; 90(1):33-37 and Donello J, etal, J. Virol. 1998; 72(6):5085-5092).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest. Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Also encompassed by the term “regulatory element”are enhancer elements. It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the host cell to be transformed, the level ofexpression desired, etc. A vector can be introduced into host cells tothereby produce transcripts, proteins, or peptides, including fusionproteins or peptides, encoded by nucleic acids as described herein(e.g., RBD).

In one embodiment, the vector contains at least one target sequence ofat least one miRNA expressed in non-tumor tissue. The term “microRNAs”or “miRNAs”, as used herein, are small (~22-nt), evolutionarilyconserved, regulatory RNAs involved in RNA-mediated gene silencing atthe post-transcriptional level (see, e.g., Barrel DP. Cell 2004; 116:281-297). Through base pairing with complementary regions (most often inthe 3′ untranslated region (3′UTR) of cellular messenger RNA (mRNA)),miRNAs can act to suppress mRNA translation or, upon high-sequencehomology, cause the catalytic degradation of mRNA. Because of the highlydifferential tissue expression of many miRNAs, cellular miRNAs can beexploited to mediate tissue-specific targeting of gene therapy vectors.By engineering tandem copies of target elements perfectly complementaryto tissue-specific miRNAs (miRT).

Antibodies

In certain embodiments, the one or more agents is an antibody. Incertain embodiments, the antibody blocks or disrupts the XPR1:KIDINS220protein complex from functioning to export inorganic phosphate from atumor cell. In example embodiments, the antibody is specific for XPR1,KIDINS220, or the XPR1/KIDINS220 protein complex. In certainembodiments, the antibody blocks the XPR1:KIDINS220 protein complex fromforming. In certain embodiments, the antibody targets XPR1 (see, e.g.,WO2020153467A1). In certain embodiments, the antibody targets anextracellular domain of XPR1. In certain embodiments, the antibodytargets the SPX domain or EXS domain of XPR1. In certain embodiments,XPR1 antibodies target the region spanning amino acid 529 to the end ofthe protein, which is commonly available from different vendors. Incertain embodiments, the antibody targets a Walker A/B motif ofKIDINS220. In certain embodiments, the antibody targets a Walker A/Bmotif or KAP family P-loop domain of KIDINS220 to block phosphatebinding.

Antibodies recognizing XPR1 or KIDINS220 have been generated and arecommercially available (see, e.g., ThermoFisher website: Cat#21856-1-AP, Cat #PA5-116475, Cat #PA5-100152, Cat #PA5-97897, Cat#PA5-22116, Cat #MA5-32869, Cat #PA5-82511, Cat #PA5-111894, Cat#66748-1-IG, Cat #A303-002A, Cat #A303-003A, Cat #MA1-90667, Cat#PA1-4229, Cat #BS-7041R, Cat #A303-002A-M, Cat #RA19019, Cat #RA19020;and Cat #PA5-82552, Cat #PA5-21908, Cat #PA5-34338, Cat #14174-1-AP, Cat#PA5-34337, Cat #PA1-23547). One skilled in the art could easilygenerate a therapeutic antibody to block XPR1 or KIDINS220 to inhibitphosphate efflux (see, e.g., Lu, RM., Hwang, YC., Liu, IJ. et al.Development of therapeutic antibodies for the treatment of diseases. JBiomed Sci 27, 1 (2020)).

The term “antibody” is used interchangeably with the term“immunoglobulin” herein, and includes intact antibodies, fragments ofantibodies, e.g., Fab, F(ab′)2 fragments, and intact antibodies andfragments that have been mutated either in their constant and/orvariable region (e.g., mutations to produce chimeric, partiallyhumanized, or fully humanized antibodies, as well as to produceantibodies with a desired trait, e.g., enhanced binding and/or reducedFcR binding). The term “fragment” refers to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain. Fragments can be obtainedvia chemical or enzymatic treatment of an intact or complete antibody orantibody chain. Fragments can also be obtained by recombinant means.Exemplary fragments include a nanobody, Fab, Fab′, (Fab′)2, Fv, ScFv,diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, or Fab3fragment, Fabc, Fd, dAb, V_(HH) and scFv and/or Fv fragments.

As used herein, a preparation of antibody protein having less than about50% of non-antibody protein (also referred to herein as a “contaminatingprotein”), or of chemical precursors, is considered to be “substantiallyfree.” 40%, 30%, 20%, 10% and more preferably 5% (by dry weight), ofnon-antibody protein, or of chemical precursors is considered to besubstantially free. When the antibody protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, i.e., culture medium representsless than about 30%, preferably less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume or mass of the protein preparation.

The term “antigen-binding fragment” refers to a polypeptide fragment ofan immunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). As such these antibodiesor fragments thereof are included in the scope of the invention,provided that the antibody or fragment binds specifically to a targetmolecule.

It is intended that the term “antibody” encompass any Ig class or any Igsubclass (e.g. the IgG1, IgG2, IgG3, and IgG4 subclassess of IgG)obtained from any source (e.g., humans and non-human primates, and inrodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

The term “Ig class” or “immunoglobulin class”, as used herein, refers tothe five classes of immunoglobulin that have been identified in humansand higher mammals, IgG, IgM, IgA, IgD, and IgE. The term “Ig subclass”refers to the two subclasses of IgM (H and L), three subclasses of IgA(IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2,IgG3, and IgG4) that have been identified in humans and higher mammals.The antibodies can exist in monomeric or polymeric form; for example,IgM antibodies exist in pentameric form, and IgA antibodies exist inmonomeric, dimeric or multimeric form.

The term “IgG subclass” refers to the four subclasses of immunoglobulinclass IgG – IgG1, IgG2, IgG3, and IgG4 that have been identified inhumans and higher mammals by the heavy chains of the immunoglobulins,V1 - γ4, respectively. The term “single-chain immunoglobulin” or“single-chain antibody” (used interchangeably herein) refers to aprotein having a two-polypeptide chain structure consisting of a heavyand a light chain, said chains being stabilized, for example, byinterchain peptide linkers, which has the ability to specifically bindantigen. The term “domain” refers to a globular region of a heavy orlight chain polypeptide comprising peptide loops (e.g., comprising 3 to4 peptide loops) stabilized, for example, by β pleated sheet and/orintrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequencevariation within the domains of various class members in the case of a“constant” domain, or the significant variation within the domains ofvarious class members in the case of a “variable” domain. Antibody orpolypeptide “domains” are often referred to interchangeably in the artas antibody or polypeptide “regions”. The “constant” domains of anantibody light chain are referred to interchangeably as “light chainconstant regions”, “light chain constant domains”, “CL” regions or “CL”domains. The “constant” domains of an antibody heavy chain are referredto interchangeably as “heavy chain constant regions”, “heavy chainconstant domains”, “CH” regions or “CH” domains). The “variable” domainsof an antibody light chain are referred to interchangeably as “lightchain variable regions”, “light chain variable domains”, “VL” regions or“VL” domains). The “variable” domains of an antibody heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavychain constant domains”, “VH” regions or “VH” domains).

The term “region” can also refer to a part or portion of an antibodychain or antibody chain domain (e.g., a part or portion of a heavy orlight chain or a part or portion of a constant or variable domain, asdefined herein), as well as more discrete parts or portions of saidchains or domains. For example, light and heavy chains or light andheavy chain variable domains include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

The term “conformation” refers to the tertiary structure of a protein orpolypeptide (e.g., an antibody, antibody chain, domain or regionthereof). For example, the phrase “light (or heavy) chain conformation”refers to the tertiary structure of a light (or heavy) chain variableregion, and the phrase “antibody conformation” or “antibody fragmentconformation” refers to the tertiary structure of an antibody orfragment thereof.

The term “antibody-like protein scaffolds” or “engineered proteinscaffolds” broadly encompasses proteinaceous non-immunoglobulinspecific-binding agents, typically obtained by combinatorial engineering(such as site-directed random mutagenesis in combination with phagedisplay or other molecular selection techniques). Usually, suchscaffolds are derived from robust and small soluble monomeric proteins(such as Kunitz inhibitors or lipocalins) or from a stably foldedextra-membrane domain of a cell surface receptor (such as protein A,fibronectin or the ankyrin repeat).

Such scaffolds have been extensively reviewed in Binz et al.(Engineering novel binding proteins from nonimmunoglobulin domains. NatBiotechnol 2005, 23:1257-1268), Gebauer and Skerra (Engineered proteinscaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol.2009, 13:245-55), Gill and Damle (Biopharmaceutical drug discovery usingnovel protein scaffolds. Curr Opin Biotechnol 2006, 17:653-658), Skerra(Engineered protein scaffolds for molecular recognition. J Mol Recognit2000, 13:167-187), and Skerra (Alternative non-antibody scaffolds formolecular recognition. Curr Opin Biotechnol 2007, 18:295-304), andinclude without limitation affibodies, based on the Z-domain ofstaphylococcal protein A, a three-helix bundle of 58 residues providingan interface on two of its alpha-helices (Nygren, Alternative bindingproteins: Affibody binding proteins developed from a small three-helixbundle scaffold. FEBS J 2008, 275:2668-2676); engineered Kunitz domainsbased on a small (ca. 58 residues) and robust, disulphide-crosslinkedserine protease inhibitor, typically of human origin (e.g., LACI-D1),which can be engineered for different protease specificities (Nixon andWood, Engineered protein inhibitors of proteases. Curr Opin Drug DiscovDev 2006, 9:261-268); monobodies or adnectins based on the 10thextracellular domain of human fibronectin III (10Fn3), which adopts anIg-like beta-sandwich fold (94 residues) with 2-3 exposed loops, butlacks the central disulphide bridge (Koide and Koide, Monobodies:antibody mimics based on the scaffold of the fibronectin type IIIdomain. Methods Mol Biol 2007, 352:95-109); anticalins derived from thelipocalins, a diverse family of eight-stranded beta-barrel proteins (ca.180 residues) that naturally form binding sites for small ligands bymeans of four structurally variable loops at the open end, which areabundant in humans, insects, and many other organisms (Skerra,Alternative binding proteins: Anticalins-harnessing the structuralplasticity of the lipocalin ligand pocket to engineer novel bindingactivities. FEBS J 2008, 275:2677-2683); DARPins, designed ankyrinrepeat domains (166 residues), which provide a rigid interface arisingfrom typically three repeated beta-turns (Stumpp et al., DARPins: a newgeneration of protein therapeutics. Drug Discov Today 2008, 13:695-701);avimers (multimerized LDLR-A module) (Silverman et al., Multivalentavimer proteins evolved by exon shuffling of a family of human receptordomains. Nat Biotechnol 2005, 23:1556-1561); and cysteine-rich knottinpeptides (Kolmar, Alternative binding proteins: biological activity andtherapeutic potential of cystine-knot miniproteins. FEBS J 2008,275:2684-2690).

“Specific binding” of an antibody means that the antibody exhibitsappreciable affinity for a particular antigen or epitope and, generally,does not exhibit significant cross reactivity. “Appreciable” bindingincludes binding with an affinity of at least 25 µM. Antibodies withaffinities greater than 1 × 10⁷ M⁻¹ (or a dissociation coefficient of1uM or less or a dissociation coefficient of 1 nm or less) typicallybind with correspondingly greater specificity. Values intermediate ofthose set forth herein are also intended to be within the scope of thepresent invention and antibodies of the invention bind with a range ofaffinities, for example, 100 nM or less, 75 nM or less, 50 nM or less,25 nM or less, for example 10 nM or less, 5 nM or less, 1 nM or less, orin embodiments 500pM or less, 100pM or less, 50pM or less or 25pM orless. An antibody that “does not exhibit significant crossreactivity” isone that will not appreciably bind to an entity other than its target(e.g., a different epitope or a different molecule). For example, anantibody that specifically binds to a target molecule will appreciablybind the target molecule but will not significantly react withnon-target molecules or peptides. An antibody specific for a particularepitope will, for example, not significantly crossreact with remoteepitopes on the same protein or peptide. Specific binding can bedetermined according to any art-recognized means for determining suchbinding. Preferably, specific binding is determined according toScatchard analysis and/or competitive binding assays.

As used herein, the term “affinity” refers to the strength of thebinding of a single antigen-combining site with an antigenicdeterminant. Affinity depends on the closeness of stereochemical fitbetween antibody combining sites and antigen determinants, on the sizeof the area of contact between them, on the distribution of charged andhydrophobic groups, etc. Antibody affinity can be measured byequilibrium dialysis or by the kinetic BIACORE™ method. The dissociationconstant, Kd, and the association constant, Ka, are quantitativemeasures of affinity.

As used herein, the term “monoclonal antibody” refers to an antibodyderived from a clonal population of antibody-producing cells (e.g., Blymphocytes or B cells) which is homogeneous in structure and antigenspecificity. The term “polyclonal antibody” refers to a plurality ofantibodies originating from different clonal populations ofantibody-producing cells which are heterogeneous in their structure andepitope specificity but which recognize a common antigen. Monoclonal andpolyclonal antibodies may exist within bodily fluids, as crudepreparations, or may be purified, as described herein.

The term “binding portion” of an antibody (or “antibody portion”)includes one or more complete domains, e.g., a pair of complete domains,as well as fragments of an antibody that retain the ability tospecifically bind to a target molecule. It has been shown that thebinding function of an antibody can be performed by fragments of afull-length antibody. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intactimmunoglobulins. Binding fragments include Fab, Fab′, F(ab′)2, Fabc, Fd,dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and singledomain antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, FR residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all or atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

Examples of portions of antibodies or epitope-binding proteinsencompassed by the present definition include: (i) the Fab fragment,having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii) the Fab′ fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the C_(H)1 domain; (iii) the Fd fragment having V_(H) andC_(H)1 domains; (iv) the Fd′ fragment having V_(H) and C_(H)1 domainsand one or more cysteine residues at the C-terminus of the CHI domain;(v) the Fv fragment having the V_(L) and V_(H) domains of a single armof an antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544(1989)) which consists of a V_(H) domain or a V_(L) domain that bindsantigen; (vii) isolated CDR regions or isolated CDR regions presented ina functional framework; (viii) F(ab′)₂ fragments which are bivalentfragments including two Fab′ fragments linked by a disulphide bridge atthe hinge region; (ix) single chain antibody molecules (e.g., singlechain Fv; scFv) (Bird et al., 242 Science 423 (1988); and Huston et al.,85 PNAS 5879 (1988)); (x) “diabodies” with two antigen binding sites,comprising a heavy chain variable domain (V_(H)) connected to a lightchain variable domain (V_(L)) in the same polypeptide chain (see, e.g.,EP 404,097; WO 93/11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi)“linear antibodies” comprising a pair of tandem Fd segments(V_(H)-C_(h)1-V_(H)-C_(h)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions (Zapata etal., Protein Eng. 8(10):1057-62 (1995); and U.S. Pat. No. 5,641,870).

As used herein, a “blocking” antibody or an antibody “antagonist” is onewhich inhibits or reduces biological activity of the antigen(s) itbinds. In certain embodiments, the blocking antibodies or antagonistantibodies or portions thereof described herein completely inhibit thebiological activity of the antigen(s).

Antibodies may act as agonists or antagonists of the recognizedpolypeptides. For example, the present invention includes antibodieswhich disrupt receptor/ligand interactions either partially or fully.The invention features both receptor-specific antibodies andligand-specific antibodies. The invention also featuresreceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. For example, receptor activation can be determined by detecting thephosphorylation (e.g., tyrosine or serine/threonine) of the receptor orof one of its down-stream substrates by immunoprecipitation followed bywestern blot analysis. In specific embodiments, antibodies are providedthat inhibit ligand activity or receptor activity by at least 95%, atleast 90%, at least 85%, at least 80%, at least 75%, at least 70%, atleast 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex. Likewise, encompassed by theinvention are neutralizing antibodies which bind the ligand and preventbinding of the ligand to the receptor, as well as antibodies which bindthe ligand, thereby preventing receptor activation, but do not preventthe ligand from binding the receptor. Further included in the inventionare antibodies which activate the receptor. These antibodies may act asreceptor agonists, i.e., potentiate or activate either all or a subsetof the biological activities of the ligand-mediated receptor activation,for example, by inducing dimerization of the receptor. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides disclosed herein. The antibody agonists and antagonists canbe made using methods known in the art. See, e.g., PCT publication WO96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988(1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al.,J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res.58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179(1998); Prat et al., J. Cell. Sci. III (Pt2):237-247 (1998); Pitard etal., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al.,Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996).

The antibodies as defined for the present invention include derivativesthat are modified, i.e., by the covalent attachment of any type ofmolecule to the antibody such that covalent attachment does not preventthe antibody from generating an anti-idiotypic response. For example,but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited tospecific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

Simple binding assays can be used to screen for or detect agents thatbind to a target protein, or disrupt the interaction between proteins(e.g., a receptor and a ligand). Because certain targets of the presentinvention are transmembrane proteins, assays that use the soluble formsof these proteins rather than full-length protein can be used, in someembodiments. Soluble forms include, for example, those lacking thetransmembrane domain and/or those comprising the IgV domain or fragmentsthereof which retain their ability to bind their cognate bindingpartners. Further, agents that inhibit or enhance protein interactionsfor use in the compositions and methods described herein, can includerecombinant peptido-mimetics.

Detection methods useful in screening assays include antibody-basedmethods, detection of a reporter moiety, detection of cytokines asdescribed herein, and detection of a gene signature as described herein.

Another variation of assays to determine binding of a receptor proteinto a ligand protein is through the use of affinity biosensor methods.Such methods may be based on the piezoelectric effect, electrochemistry,or optical methods, such as ellipsometry, optical wave guidance, andsurface plasmon resonance (SPR).

Therapeutic Polypeptide Modifications

In certain example embodiments, the therapeutic polypeptides of thepresent invention may be modified, such that they acquire advantageousproperties for therapeutic use (e.g., stability and specificity), butmaintain their biological activity. Therapeutic proteins may be modifiedto increase stability or to provide characteristics that improveefficacy of the protein when administered to a subject in vivo. As usedherein in reference to therapeutic proteins, the terms “modified”,“modification” and the like refer to one or more changes that enhance adesired property of the therapeutic protein, where the change does notalter the primary amino acid sequence of the therapeutic protein.“Modification” includes a covalent chemical modification that does notalter the primary amino acid sequence of the therapeutic protein itself.Such desired properties include, for example, prolonging the in vivohalf-life, increasing the stability, reducing the clearance, alteringthe immunogenicity or allergenicity, enabling the raising of particularantibodies, or cellular targeting. Changes to a therapeutic protein thatmay be carried out include, but are not limited to, conjugation to acarrier protein, conjugation to a ligand, conjugation to an antibody,PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fcfusion, albumin fusion, nanoparticle attachment, nanoparticulateencapsulation, cholesterol fusion, iron fusion, acylation, amidation,glycosylation, side chain oxidation, phosphorylation, biotinylation, theaddition of a surface active material, the addition of amino acidmimetics, or the addition of unnatural amino acids. Modified therapeuticproteins also include analogs. By “analog” is meant a molecule that isnot identical, but has analogous functional or structural features. Forexample, a therapeutic protein analog retains the biological activity ofa corresponding naturally-occurring polypeptide, while having certainbiochemical modifications that enhance the analog’s function relative toa naturally-occurring polypeptide. Such biochemical modifications couldincrease the analog’s protease resistance, membrane permeability, orhalf-life, without altering, for example, ligand binding. An analog mayinclude an unnatural amino acid.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Modified proteins may include a spacer or a linker. The terms “spacer”or “linker” as used in reference to a fusion protein refers to a peptidethat joins the proteins comprising a fusion protein. Generally, a spacerhas no specific biological activity other than to join or to preservesome minimum distance or other spatial relationship between theproteins. However, in certain embodiments, the constituent amino acidsof a spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity of the molecule.

Suitable linkers for use in an embodiment of the present invention arewell known to those of skill in the art and include, but are not limitedto, straight or branched-chain carbon linkers, heterocyclic carbonlinkers, or peptide linkers. The linker is used to separate two peptidesby a distance sufficient to ensure that, in a preferred embodiment, eachpeptide properly folds. Preferred peptide linker sequences adopt aflexible extended conformation and do not exhibit a propensity fordeveloping an ordered secondary structure. Typical amino acids inflexible protein regions include Gly, Asn and Ser. Virtually anypermutation of amino acid sequences containing Gly, Asn and Ser would beexpected to satisfy the above criteria for a linker sequence. Other nearneutral amino acids, such as Thr and Ala, also may be used in the linkersequence. Still other amino acid sequences that may be used as linkersare disclosed in Maratea et al. (1985), Gene 40: 39-46; Murphy et al.(1986) Proc. Nat′l. Acad. Sci. USA 83 : 8258-62; U.S. Pat. No.4,935,233; and U.S. Pat. No. 4,751, 180.

The clinical effectiveness of protein therapeutics is often limited byshort plasma half- life and susceptibility to protease degradation.Studies of various therapeutic proteins (e.g., filgrastim) have shownthat such difficulties may be overcome by various modifications,including conjugating or linking the polypeptide sequence to any of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes (see, for example, typicallyvia a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG).

It is well known that the properties of certain proteins can bemodulated by attachment of polyethylene glycol (PEG) polymers, whichincreases the hydrodynamic volume of the protein and thereby slows itsclearance by kidney filtration. (See, e.g., Clark et al., J. Biol. Chem.271: 21969-21977 (1996)). Such PEG- conjugated biomolecules have beenshown to possess clinically useful properties, including better physicaland thermal stability, protection against susceptibility to enzymaticdegradation, increased solubility, longer in vivo circulating half-lifeand decreased clearance, reduced immunogenicity and antigenicity, andreduced toxicity. Therefore, it is envisioned that certain agents can bePEGylated (e.g., on peptide residues) to provide enhanced therapeuticbenefits such as, for example, increased efficacy by extending half-lifein vivo. In certain embodiments, PEGylation of the agents may be used toextend the serum half-life of the agents and allow for particular agentsto be capable of crossing the blood-brain barrier. Thus, in oneembodiment, PEGylating XPR1:KIDINS220 antagonists improve thepharmacokinetics and pharmacodynamics of the antagonists.

In regard to peptide PEGylation methods, reference is made to Lu et al.,Int. J. Pept. Protein Res.43: 127-38 (1994); Lu et al., Pept. Res. 6:140-6 (1993); Felix et al., Int. J. Pept. Protein Res. 46: 253-64(1995); Gaertner et al., Bioconjug. Chem. 7: 38-44 (1996); Tsutsumi etal., Thromb. Haemost. 77: 168-73 (1997); Francis et al., hit. J.Hematol. 68: 1-18 (1998); Roberts et al., J. Pharm. Sci. 87: 1440-45(1998); and Tan et al., Protein Expr. Purif. 12: 45-52 (1998).Polyethylene glycol or PEG is meant to encompass any of the forms of PEGthat have been used to derivatize other proteins, including, but notlimited to, mono-(C1-10) alkoxy or aryloxy-polyethylene glycol. SuitablePEG moieties include, for example, 40 kDa methoxy poly(ethylene glycol)propionaldehyde (Dow, Midland, Mich.); 60 kDa methoxy poly(ethyleneglycol) propionaldehyde (Dow, Midland, Mich.); 40 kDa methoxypoly(ethylene glycol) maleimido-propionamide (Dow, Midland, Mich.); 31kDa alpha-methyl-w-(3-oxopropoxy), polyoxyethylene (NOF Corporation,Tokyo); mPEG2-NHS-40k (Nektar); mPEG2-MAL-40k (Nektar), SUNBRIGHTGL2-400MA ((PEG)240kDa) (NOF Corporation, Tokyo), SUNBRIGHT ME-200MA(PEG20kDa) (NOF Corporation, Tokyo). The PEG groups are generallyattached to the peptide (e.g., RBD) via acylation or alkylation througha reactive group on the PEG moiety (for example, a maleimide, analdehyde, amino, thiol, or ester group) to a reactive group on thepeptide (for example, an aldehyde, amino, thiol, a maleimide, or estergroup).

The PEG molecule(s) may be covalently attached to any Lys, Cys, orK(CO(CH2)2SH) residues at any position in a peptide. In certainembodiments, the RBD proteins described herein can be PEGylated directlyto any amino acid at the N-terminus by way of the N-terminal aminogroup. A “linker arm” may be added to a peptide to facilitatePEGylation. PEGylation at the thiol side-chain of cysteine has beenwidely reported (see, e.g., Caliceti & Veronese, Adv. Drug Deliv. Rev.55: 1261-77 (2003)). If there is no cysteine residue in the peptide, acysteine residue can be introduced through substitution or by adding acysteine to the N-terminal amino acid. In certain embodiments, proteinsare PEGylated through the side chains of a cysteine residue added to theN-terminal amino acid.

In exemplary embodiments, the PEG molecule(s) may be covalently attachedto an amide group in the C-terminus of a peptide, such as in the RBDprotein. In certain embodiments, the PEG molecule used in modifying anagent of the present invention is branched while in other embodiments,the PEG molecule may be linear. In particular aspects, the PEG moleculeis between 1 kDa and 100 kDa in molecular weight. In further aspects,the PEG molecule is selected from 10, 20, 30, 40, 50, 60, and 80 kDa. Infurther still aspects, it is selected from 20, 40, or 60 kDa. Wherethere are two PEG molecules covalently attached to the agent of thepresent invention, each is 1 to 40 kDa and in particular aspects, theyhave molecular weights of 20 and 20 kDa, 10 and 30 kDa, 30 and 30 kDa,20 and 40 kDa, or 40 and 40 kDa. In particular aspects, the agent (e.g.,XPR1:KIDINS220 antagonists) contain mPEG-cysteine. The mPEG inmPEG-cysteine can have various molecular weights. The range of themolecular weight is preferably 5 kDa to 200 kDa, more preferably 5 kDato 100 kDa, and further preferably 20 kDa to 60 kDA. The mPEG can belinear or branched.

The present disclosure also contemplates the use of PEG Mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half- life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the therapeutic protein (e.g.,Amunix’ XTEN technology; Mountain View, CA). This obviates the need foran additional conjugation step during the manufacturing process.Moreover, established molecular biology techniques enable control of theside chain composition of the polypeptide chains, allowing optimizationof immunogenicity and manufacturing properties.

Glycosylation can dramatically affect the physical properties ofproteins and can also be important in protein stability, secretion, andsubcellular localization (see, e.g., Solá and Griebenow, Glycosylationof Therapeutic Proteins: An Effective Strategy to Optimize Efficacy.BioDrugs. 2010; 24(1): 9-21). Proper glycosylation can be essential forbiological activity. In fact, some genes from eukaryotic organisms, whenexpressed in bacteria (e.g., E. coli) which lack cellular processes forglycosylating proteins, yield proteins that are recovered with little orno activity by virtue of their lack of glycosylation. For purposes ofthe present disclosure, “glycosylation” is meant to broadly refer to theenzymatic process that attaches glycans to proteins, lipids or otherorganic molecules. The use of the term “glycosylation” in conjunctionwith the present disclosure is generally intended to mean adding ordeleting one or more carbohydrate moieties (either by removing theunderlying glycosylation site or by deleting the glycosylation bychemical and/or enzymatic means), and/or adding one or moreglycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence. The alteration to the polypeptide may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for O-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. A particular embodiment of thepresent disclosure comprises the generation and use of N-glycosylationvariants.

The present disclosure also contemplates the use of polysialylation, theconjugation of peptides and proteins to the naturally occurring,biodegradable a-(2→8) linked polysialic acid (“PSA”) in order to improvetheir stability and in vivo pharmacokinetics. PSA is a biodegradable,non-toxic natural polymer that is highly hydrophilic, giving it a highapparent molecular weight in the blood which increases its serumhalf-life. In addition, polysialylation of a range of peptide andprotein therapeutics has led to markedly reduced proteolysis, retentionof activity in vivo activity, and reduction in immunogenicity andantigenicity (see, e.g., G. Gregoriadis et al., Int. J. Pharmaceutics300(1-2): 125-30). As with modifications with other conjugates (e.g.,PEG), various techniques for site-specific polysialylation are available(see, e.g., T. Lindhout et al., PNAS 108(18)7397-7402 (2011)).

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; albumins such as human serum albumin (HAS);tetanus toxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemaglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Fusion of albumin to one or more polypeptides of the present disclosurecan, for example, be achieved by genetic manipulation, such that the DNAcoding for HSA, or a fragment thereof, is joined to the DNA coding forthe one or more polypeptide sequences. Albumin itself may be modified toextend its circulating half-life. Fusion of the modified albumin to oneor more Polypeptides can be attained by the genetic manipulationtechniques described above or by chemical conjugation; the resultingfusion molecule has a half- life that exceeds that of fusions withnon-modified albumin. (See WO2011/051489).

Several albumin — binding strategies have been developed as alternativesfor direct fusion, including albumin binding through a conjugated fattyacid chain (acylation). Because serum albumin is a transport protein forfatty acids, these natural ligands with albumin — binding activity havebeen used for half-life extension of small protein therapeutics. Forexample, insulin determir (LEVEMIR), an approved product for diabetes,comprises a myristyl chain conjugated to a genetically-modified insulin,resulting in a long- acting insulin analog.

Another type of modification is to conjugate (e.g., link) one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another protein, or a carrier molecule.Thus, an exemplary polypeptide sequence can be provided as a conjugatewith another component or molecule. A conjugate modification may resultin a polypeptide sequence that retains activity with an additional orcomplementary function or activity of the second molecule. For example,a polypeptide sequence may be conjugated to a molecule, e.g., tofacilitate solubility, storage, in vivo or shelf half-life or stability,reduction in immunogenicity, delayed or controlled release in vivo, etc.Other functions or activities include a conjugate that reduces toxicityrelative to an unconjugated polypeptide sequence, a conjugate thattargets a type of cell or organ more efficiently than an unconjugatedpolypeptide sequence, or a drug to further counter the causes or effectsassociated with a disorder or disease as set forth herein.

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of the Polypeptides toimprove one or more properties. One such method for prolonging thecirculation half-life, increasing the stability, reducing the clearance,or altering the immunogenicity or allergenicity of a polypeptide of thepresent disclosure involves modification of the polypeptide sequences byhesylation, which utilizes hydroxyethyl starch derivatives linked toother molecules in order to modify the molecule’s characteristics.Various aspects of hesylation are described in, for example, U.S. Pat.Appln. Nos. 2007/0134197 and 2006/0258607.

In particular embodiments, the agents (e.g., XPR1:KIDINS220 antagonists,RBD) include a protecting group covalently joined to the N-terminalamino group. In exemplary embodiments, a protecting group covalentlyjoined to the N-terminal amino group of the proteins reduces thereactivity of the amino terminus under in vivo conditions. Aminoprotecting groups include —C1-10 alkyl, —C1-10 substituted alkyl, —C2-10alkenyl, —C2-10 substituted alkenyl, aryl, —C1-6 alkyl aryl,—C(O)—(CH2)1-6—COOH, —C(O)—C1-6 alkyl, -C(O)-aryl, —C(O)—O—C1-6 alkyl,or —C(O)—O-aryl. In particular embodiments, the amino terminusprotecting group is selected from the group consisting of acetyl,propyl, succinyl, benzyl, benzyloxycarbonyl, and t-butyloxycarbonyl. Inother embodiments, deamination of the N-terminal amino acid is anothermodification that may be used for reducing the reactivity of the aminoterminus under in vivo conditions.

Chemically modified compositions of the agents (e.g., XPR1:KIDINS220antagonists, RBD) wherein the agent is linked to a polymer are alsoincluded within the scope of the present invention. The polymer selectedis usually modified to have a single reactive group, such as an activeester for acylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled. Included within the scope of polymersis a mixture of polymers. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable. The polymer or mixture thereof may include but is notlimited to polyethylene glycol (PEG), monomethoxy-polyethylene glycol,dextran, cellulose, or other carbohydrate-based polymers, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(for example, glycerol), and polyvinyl alcohol.

In other embodiments, the agents (e.g., XPR1:KIDINS220 antagonists, RBD)are modified by PEGylation, cholesterylation, or palmitoylation. Themodification can be to any amino acid residue. In preferred embodiments,the modification is to the N-terminal amino acid of the agent (e.g.,XPR1:KIDINS220 antagonists, RBD), either directly to the N-terminalamino acid or by way coupling to the thiol group of a cysteine residueadded to the N-terminus or a linker added to the N-terminus such astrimesoyl tris(3,5- dibromosalicylate (Ttds). In certain embodiments,the N-terminus of the agent (e.g., XPR1:KIDINS220 antagonists, RBD)comprises a cysteine residue to which a protecting group is coupled tothe N-terminal amino group of the cysteine residue and the cysteinethiolate group is derivatized with N-ethylmaleimide, PEG group,cholesterol group, or palmitoyl group. In other embodiments, anacetylated cysteine residue is added to the N-terminus of the agents,and the thiol group of the cysteine is derivatized withN-ethylmaleimide, PEG group, cholesterol group, or palmitoyl group. Incertain embodiments, the agent of the present invention is a conjugate.In certain embodiments, the agent of the present invention is apolypeptide consisting of an amino acid sequence which is bound with amethoxypolyethylene glycol(s) via a linker.

Substitutions of amino acids may be used to modify an agent of thepresent invention. The phrase “substitution of amino acids” as usedherein encompasses substitution of amino acids that are the result ofboth conservative and non-conservative substitutions. Conservativesubstitutions are the replacement of an amino acid residue by anothersimilar residue in a polypeptide. Typical but not limiting conservativesubstitutions are the replacements, for one another, among the aliphaticamino acids Ala, Val, Leu and Ile; interchange of Ser and Thr containinghydroxy residues, interchange of the acidic residues Asp and Glu,interchange between the amide-containing residues Asn and Gln,interchange of the basic residues Lys and Arg, interchange of thearomatic residues Phe and Tyr, and interchange of the small-sized aminoacids Ala, Ser, Thr, Met, and Gly. Non-conservative substitutions arethe replacement, in a polypeptide, of an amino acid residue by anotherresidue which is not biologically similar. For example, the replacementof an amino acid residue with another residue that has a substantiallydifferent charge, a substantially different hydrophobicity, or asubstantially different spatial configuration.

One of skill in the art from this disclosure and the knowledge in theart will appreciate that there are a variety of ways in which to producesuch therapeutic proteins. In general, such therapeutic proteins may beproduced either in vitro or in vivo. Therapeutic proteins may beproduced in vitro as peptides or polypeptides, which may then beformulated into a pharmaceutical composition and administered to asubject. Such in vitro production may occur by a variety of methodsknown to one of skill in the art such as, for example, peptide synthesisor expression of a peptide/polypeptide from a DNA or RNA molecule in anyof a variety of bacterial, eukaryotic, or viral recombinant expressionsystems, followed by purification of the expressed peptide/polypeptide.Alternatively, therapeutic proteins may be produced in vivo byintroducing molecules (e.g., DNA, RNA, viral expression systems, and thelike) that encode therapeutic proteins into a subject, whereupon theencoded therapeutic proteins are expressed.

Small Molecules

In certain embodiments, the one or more therapeutic agents comprise asmall molecule that inhibits expression of XPR1, KINDINS220, inhibitsformation of the XPR1:KINDINS220 complex, or inhibits phosphate effluxby the XPR1:KINDINS220 complex. The term “small molecule” refers tocompounds, preferably organic compounds, with a size comparable to thoseorganic molecules generally used in pharmaceuticals. The term excludesbiological macromolecules (e.g., proteins, peptides, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, e.g., up to about 4000, preferably up to 3000 Da, more preferably upto 2000 Da, even more preferably up to about 1000 Da, e.g., up to about900, 800, 700, 600 or up to about 500 Da. In certain embodiments, thesmall molecule may act as an antagonist or agonist (e.g., blocking anenzyme active site or activating a receptor by binding to a ligandbinding site). In certain embodiments, the small molecule blocks ordisrupts the XPR1:KIDINS220 protein complex from functioning to exportinorganic phosphate from a tumor cell. In certain embodiments, the smallmolecule blocks the XPR1:KIDINS220 protein complex from forming.

In one example embodiment, a small molecule blocks a phosphate bindingdomain, such as the Walker A/B motif of KIDINS220 (see, e.g., SandallCF, Ziehr BK, MacDonald JA. ATP-Binding and Hydrolysis in InflammasomeActivation. Molecules. 2020;25(19):4572). MCC950 is adiarylsulfonylurea-containing compound that suppresses ATP hydrolysisthrough direct binding of the ATP binding region of NLRP3, likely withinthe Walker B motif (see, e.g., Coll RC, Hill JR, Day CJ, et al. MCC950directly targets the NLRP3 ATP-hydrolysis motif for inflammasomeinhibition. Nat Chem Biol. 2019;15(6):556-559).

One type of small molecule applicable to the present invention is adegrader molecule (see, e.g., Ding, et al., Emerging New Concepts ofDegrader Technologies, Trends Pharmacol Sci. 2020 Jul;41(7):464-474).The terms “degrader” and “degrader molecule” refer to all compoundscapable of specifically targeting a protein for degradation (e.g.,ATTEC, AUTAC, LYTAC, or PROTAC, reviewed in Ding, et al. 2020).Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emergingalternative therapeutic strategy with the potential to address many ofthe challenges currently faced in modern drug development programs.PROTAC technology employs small molecules that recruit target proteinsfor ubiquitination and removal by the proteasome (see, e.g., Zhou etal., Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable ofAchieving Tumor Regression. J. Med. Chem. 2018, 61, 462-481; Bondesonand Crews, Targeted Protein Degradation by Small Molecules, Annu RevPharmacol Toxicol. 2017 Jan 6; 57: 107-123; and Lai et al., ModularPROTAC Design for the Degradation of Oncogenic BCR-ABL Angew Chem Int EdEngl. 2016 Jan 11; 55(2): 807-810). In certain embodiments, LYTACs areparticularly advantageous for cell surface proteins as described herein(e.g., XPR1 and/or KIDINS220).

Aptamers

In certain embodiments, the one or more agents is an aptamer. Nucleicacid aptamers are nucleic acid species that have been engineered throughrepeated rounds of in vitro selection or equivalently, SELEX (systematicevolution of ligands by exponential enrichment) to bind to variousmolecular targets such as small molecules, proteins, nucleic acids,cells, tissues and organisms. Nucleic acid aptamers have specificbinding affinity to molecules through interactions other than classicWatson-Crick base pairing. Aptamers are useful in biotechnological andtherapeutic applications as they offer molecular recognition propertiessimilar to antibodies. In addition to their discriminate recognition,aptamers offer advantages over antibodies as they can be engineeredcompletely in a test tube, are readily produced by chemical synthesis,possess desirable storage properties, and elicit little or noimmunogenicity in therapeutic applications. In certain embodiments, RNAaptamers may be expressed from a DNA construct. In other embodiments, anucleic acid aptamer may be linked to another polynucleotide sequence.The polynucleotide sequence may be a double stranded DNA polynucleotidesequence. The aptamer may be covalently linked to one strand of thepolynucleotide sequence. The aptamer may be ligated to thepolynucleotide sequence. The polynucleotide sequence may be configured,such that the polynucleotide sequence may be linked to a solid supportor ligated to another polynucleotide sequence.

Aptamers, like peptides generated by phage display or monoclonalantibodies (“mAbs”), are capable of specifically binding to selectedtargets and modulating the target’s activity, e.g., through binding,aptamers may block their target’s ability to function. A typical aptameris 10-15 kDa in size (30-45 nucleotides), binds its target withsub-nanomolar affinity, and discriminates against closely relatedtargets (e.g., aptamers will typically not bind other proteins from thesame gene family). Structural studies have shown that aptamers arecapable of using the same types of binding interactions (e.g., hydrogenbonding, electrostatic complementarity, hydrophobic contacts, stericexclusion) that drives affinity and specificity in antibody-antigencomplexes.

Aptamers have a number of desirable characteristics for use in researchand as therapeutics and diagnostics including high specificity andaffinity, biological efficacy, and excellent pharmacokinetic properties.In addition, they offer specific competitive advantages over antibodiesand other protein biologics. Aptamers are chemically synthesized and arereadily scaled as needed to meet production demand for research,diagnostic or therapeutic applications. Aptamers are chemically robust.They are intrinsically adapted to regain activity following exposure tofactors such as heat and denaturants and can be stored for extendedperiods (>1 yr) at room temperature as lyophilized powders. Not beingbound by a theory, aptamers bound to a solid support or beads may bestored for extended periods.

Oligonucleotides in their phosphodiester form may be quickly degraded byintracellular and extracellular enzymes such as endonucleases andexonucleases. Aptamers can include modified nucleotides conferringimproved characteristics on the ligand, such as improved in vivostability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions. SELEX identified nucleic acid ligandscontaining modified nucleotides are described, e.g., in U.S. Pat. No.5,660,985, which describes oligonucleotides containing nucleotidederivatives chemically modified at the 2′ position of ribose, 5 positionof pyrimidines, and 8 position of purines, U.S. Pat. No. 5,756,703 whichdescribes oligonucleotides containing various 2′ -modified pyrimidines,and U.S. Pat. No. 5,580,737 which describes highly specific nucleic acidligands containing one or more nucleotides modified with 2′-amino(2′-NH₂), 2′-fluoro (2′-F), and/or 2′-0-methyl (2′-OMe) substituents.Modifications of aptamers may also include, modifications at exocyclicamines, substitution of 4- thiouridine, substitution of 5-bromo or5-iodo-uracil; backbone modifications, phosphorothioate or allylphosphate modifications, methylations, and unusual base-pairingcombinations such as the isobases isocytidine and isoguanosine.Modifications can also include 3′ and 5′ modifications such as capping.As used herein, the term phosphorothioate encompasses one or morenon-bridging oxygen atoms in a phosphodiester bond replaced by one ormore sulfur atoms. In further embodiments, the oligonucleotides comprisemodified sugar groups, for example, one or more of the hydroxyl groupsis replaced with halogen, aliphatic groups, or functionalized as ethersor amines. In one embodiment, the 2′-position of the furanose residue issubstituted by any of an O-methyl, O-alkyl, O-allyl, S-alkyl, S-allyl,or halo group. Methods of synthesis of 2′-modified sugars are described,e.g., in Sproat, et al., Nucl. Acid Res. 19:733-738 (1991); Cotten, etal, Nucl. Acid Res. 19:2629-2635 (1991); and Hobbs, et al, Biochemistry12:5138-5145 (1973). Other modifications are known to one of ordinaryskill in the art. In certain embodiments, aptamers include aptamers withimproved off-rates as described in International Patent Publication No.WO 2009012418, “Method for generating aptamers with improved off-rates,”incorporated herein by reference in its entirety. In certain embodimentsaptamers are chosen from a library of aptamers. Such libraries include,but are not limited to, those described in Rohloff et al., “Nucleic AcidLigands With Protein-like Side Chains: Modified Aptamers and Their Useas Diagnostic and Therapeutic Agents,” Molecular Therapy Nucleic Acids(2014) 3, e201. Aptamers are also commercially available (see, e.g.,SomaLogic, Inc., Boulder, Colorado). In certain embodiments, the presentinvention may utilize any aptamer containing any modification asdescribed herein.

Programmable Genetic Modifying Agents

In certain example embodiments, a programmable nuclease may be used toedit a genomic region containing XPR1 or KIDINS220. Programmable geneticmodifying agents are enzymes capable of being engineered to bind aspecific target sequences. Example programmable genetic modifying agentsinclude zinc finger nucleases, TALE nucleases (TALENS), meganucleases,and CRISPR-Cas systems. In the context of the present invention,programmable genetic modifying agents may be designed to target and/ormodify genomic DNA or mRNA of XPR1 and/or KIDINS220. The modificationsmay reduce expression of XPR1 and/or KIDINS220, or may introducestructural or post-translations modifications that inhibitXPR1:KIDINS220 complex formation. Not being bound by a theory, normalcells are not vulnerable to XPR1:KIDINS220-mediated phosphate exportinhibition, however, it is advantageous to temporarily inhibitXPR1:KIDINS220-mediated phosphate export. Thus, in certain embodiments,mRNA is targeted (e.g., RNA base editing).

CRISPR-Cas Modification

CRISPR-Cas systems comprise an endonuclease (Cas protein) capable offorming a complex with a guide molecule. The guide molecule can beengineered to comprise a sequence complementary to a given targetsequence (e.g., a target sequence within a region of XPR1 or KIDINS220).The guide molecule guides the complex to the target site where the Casendonuclease introduce a single or double-stranded cut in the targetsequence. Native cellular repair pathways, NHEJ and HDR, are used torepair the gut. NHEJ may introduce insertions or deletions at the cutsite. Accordingly, CRISPR-Cas systems can be designed to introduceinsertions or deletions that reduce or eliminate expression or interferewith XPR1/KINDS220 complex formation. Alternatively, template moleculescan be delivered with CRISPR-Cas systems that utilize the HDR pathway tointroduce insertions of desired template sequences. These insertions mayintroduce one or more mutations that reduce or eliminate expression orinterfere with XPR1/KINDS220 complex formation. The insertions mayremove or introduce post-translation modification sites, introducepremature stop codons, or disrupt splice sites that result in proteinproducts with loss of function or reduced function. CRISPR-Cas systemsmay also be modified to work with additional functional domains. In suchembodiments, the endonuclease activity of the Cas protein is eliminatedto create a dead Cas (dCas). The dCas9 is then fused with a functionaldomain. The dCas-guide complex directs the functional domain to thetarget sequence, where the functional domain introduces a modificationto a DNA or RNA target sequence. Modified CRISPR-Cas systems include DNAand RNA base editors, primer editors, and CRISPR associated transposase(CAST) systems, which are described in further detail below.

In general, a CRISPR-Cas or CRISPR system as used herein and in otherdocuments, such as WO 2014/093622 (PCT/US2013/074667), referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g., tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g., CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). See, e.g, Shmakov et al. (2015) “Discovery and FunctionalCharacterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell,DOI: dx.doi.org/10.1016/;.molcel.2015.10.008.

CRISPR-Cas systems can generally fall into two classes based on theirarchitectures of their effector molecules, which are each furthersubdivided by type and subtype. The two class are Class 1 and Class 2.Class 1 CRISPR-Cas systems have effector modules composed of multipleCas proteins, some of which form crRNA-binding complexes, while Class 2CRISPR-Cas systems include a single, multi-domain crRNA-binding protein.

In some embodiments, the CRISPR-Cas system that can be used to modify apolynucleotide of the present invention described herein can be a Class1 CRISPR-Cas system. In some embodiments, the CRISPR-Cas system that canbe used to modify a polynucleotide of the present invention describedherein can be a Class 2 CRISPR-Cas system.

Class 1 CRISPR-Cas Systems

In some embodiments, the CRISPR-Cas system that can be used to modify apolynucleotide of the present invention described herein can be a Class1 CRISPR-Cas system. Class 1 CRISPR-Cas systems are divided into typesI, II, and IV. Makarova et al. 2020. Nat. Rev. 18: 67-83., particularlyas described in FIGS. 1 . Type I CRISPR-Cas systems are divided into 9subtypes (I-A, I-B, I-C, I-D, I-E, I-F1, I-F2, I-F3, and IG). Makarovaet al., 2020. Class 1, Type I CRISPR-Cas systems can contain a Cas3protein that can have helicase activity. Type III CRISPR-Cas systems aredivided into 6 subtypes (III-A, III-B, III-C, III-D, III-E, and III-F).Type III CRISPR-Cas systems can contain a Cas10 that can include an RNArecognition motif called Palm and a cyclase domain that can cleavepolynucleotides. Makarova et al., 2020. Type IV CRISPR-Cas systems aredivided into 3 subtypes. (IV-A, IV-B, and IV-C). Makarova et al., 2020.Class 1 systems also include CRISPR-Cas variants, including Type I-A,I-B, I-E, I-F and I-U variants, which can include variants carried bytransposons and plasmids, including versions of subtype I-F encoded by alarge family of Tn7-like transposon and smaller groups of Tn7-liketransposons that encode similarly degraded subtype I-B systems. Peterset al., PNAS 114 (35) (2017); DOI: 10.1073/pnas.1709035114; see also,Makarova et al. 2018. The CRISPR Journal, v. 1, n5, FIGS. 5 .

The Class 1 systems typically use a multi-protein effector complex,which can, in some embodiments, include ancillary proteins, such as oneor more proteins in a complex referred to as a CRISPR-associated complexfor antiviral defense (Cascade), one or more adaptation proteins (e.g.,Cas1, Cas2, RNA nuclease), and/or one or more accessory proteins (e.g.,Cas 4, DNA nuclease), CRISPR associated Rossman fold (CARF) domaincontaining proteins, and/or RNA transcriptase.

The backbone of the Class 1 CRISPR-Cas system effector complexes can beformed by RNA recognition motif domain-containing protein(s) of therepeat-associated mysterious proteins (RAMPs) family subunits (e.g., Cas5, Cas6, and/or Cas7). RAMP proteins are characterized by having one ormore RNA recognition motif domains. In some embodiments, multiple copiesof RAMPs can be present. In some embodiments, the Class I CRISPR-Cassystem can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more Cas5,Cas6, and/or Cas 7 proteins. In some embodiments, the Cas6 protein is anRNAse, which can be responsible for pre-crRNA processing. When presentin a Class 1 CRISPR-Cas system, Cas6 can be optionally physicallyassociated with the effector complex.

Class 1 CRISPR-Cas system effector complexes can, in some embodiments,also include a large subunit. The large subunit can be composed of orinclude a Cas8 and/or Cas10 protein. See, e.g., FIGS. 1 and 2 . KooninEV, Makarova KS. 2019. Phil. Trans. R. Soc. B 374: 20180087, DOI:10.1098/rstb.2018.0087 and Makarova et al. 2020.

Class 1 CRISPR-Cas system effector complexes can, in some embodiments,include a small subunit (for example, Cas 11). See, e.g., FIGS. 1 and 2. Koonin EV, Makarova KS. 2019 Origins and Evolution of CRISPR-Cassystems. Phil. Trans. R. Soc. B 374: 20180087, DOI:10.1098/rstb.2018.0087.

In some embodiments, the Class 1 CRISPR-Cas system can be a Type ICRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a subtype I-A CRISPR-Cas system. In some embodiments, the Type ICRISPR-Cas system can be a subtype I-B CRISPR-Cas system. In someembodiments, the Type I CRISPR-Cas system can be a subtype I-CCRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a subtype I-D CRISPR-Cas system. In some embodiments, the Type ICRISPR-Cas system can be a subtype I-E CRISPR-Cas system. In someembodiments, the Type I CRISPR-Cas system can be a subtype I-F1CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a subtype I-F2 CRISPR-Cas system. In some embodiments, the Type ICRISPR-Cas system can be a subtype I-F3 CRISPR-Cas system. In someembodiments, the Type I CRISPR-Cas system can be a subtype I-GCRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system canbe a CRISPR Cas variant, such as a Type I-A, I-B, I-E, I-F and I-Uvariants, which can include variants carried by transposons andplasmids, including versions of subtype I-F encoded by a large family ofTn7-like transposon and smaller groups of Tn7-like transposons thatencode similarly degraded subtype I-B systems as previously described.

In some embodiments, the Class 1 CRISPR-Cas system can be a Type IIICRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas systemcan be a subtype III-A CRISPR-Cas system. In some embodiments, the TypeIII CRISPR-Cas system can be a subtype III-B CRISPR-Cas system. In someembodiments, the Type III CRISPR-Cas system can be a subtype III-CCRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas systemcan be a subtype III-D CRISPR-Cas system. In some embodiments, the TypeIII CRISPR-Cas system can be a subtype III-E CRISPR-Cas system. In someembodiments, the Type III CRISPR-Cas system can be a subtype III-FCRISPR-Cas system.

In some embodiments, the Class 1 CRISPR-Cas system can be a Type IVCRISPR-Cas-system. In some embodiments, the Type IV CRISPR-Cas systemcan be a subtype IV-A CRISPR-Cas system. In some embodiments, the TypeIV CRISPR-Cas system can be a subtype IV-B CRISPR-Cas system. In someembodiments, the Type IV CRISPR-Cas system can be a subtype IV-CCRISPR-Cas system.

The effector complex of a Class 1 CRISPR-Cas system can, in someembodiments, include a Cas3 protein that is optionally fused to a Cas2protein, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas10, a Cas11, or acombination thereof. In some embodiments, the effector complex of aClass 1 CRISPR-Cas system can have multiple copies, such as 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of any one or more Cas proteins.

Class 2 CRISPR-Cas Systems

The compositions, systems, and methods described in greater detailelsewhere herein can be designed and adapted for use with Class 2CRISPR-Cas systems. Thus, in some embodiments, the CRISPR-Cas system isa Class 2 CRISPR-Cas system. Class 2 systems are distinguished fromClass 1 systems in that they have a single, large, multi-domain effectorprotein. In certain example embodiments, the Class 2 system can be aType II, Type V, or Type VI system, which are described in Makarova etal. “Evolutionary classification of CRISPR-Cas systems: a burst of class2 and derived variants” Nature Reviews Microbiology, 18:67-81 (February2020), incorporated herein by reference. Each type of Class 2 system isfurther divided into subtypes. See Markova et al. 2020, particularly atFIGS. 2 . Class 2, Type II systems can be divided into 4 subtypes: II-A,II-B, II-C1, and II-C2. Class 2, Type V systems can be divided into 17subtypes: V-A, V-B1, V-B2, V-C, V-D, V-E, V-F1, V-F1(V-U3), V-F2, V-F3,V-G, V-H, V-I, V-K (V-U5), V-U1, V-U2, and V-U4. Class 2, Type IVsystems can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, andVI-D.

The distinguishing feature of these types is that their effectorcomplexes consist of a single, large, multi-domain protein. Type Vsystems differ from Type II effectors (e.g., Cas9), which contain twonuclear domains that are each responsible for the cleavage of one strandof the target DNA, with the HNH nuclease inserted inside the Ruv-C likenuclease domain sequence. The Type V systems (e.g., Cas12) only containa RuvC-like nuclease domain that cleaves both strands. Type VI (Cas13)are unrelated to the effectors of Type II and V systems and contain twoHEPN domains and target RNA. Cas13 proteins also display collateralactivity that is triggered by target recognition. Some Type V systemshave also been found to possess this collateral activity with twosingle-stranded DNA in in vitro contexts.

In some embodiments, the Class 2 system is a Type II system. In someembodiments, the Type II CRISPR-Cas system is a II-A CRISPR-Cas system.In some embodiments, the Type II CRISPR-Cas system is a II-B CRISPR-Cassystem. In some embodiments, the Type II CRISPR-Cas system is a II-C1CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system isa II-C2 CRISPR-Cas system. In some embodiments, the Type II system is aCas9 system. In some embodiments, the Type II system includes a Cas9.

In some embodiments, the Class 2 system is a Type V system. In someembodiments, the Type V CRISPR-Cas system is a V-A CRISPR-Cas system. Insome embodiments, the Type V CRISPR-Cas system is a V-B1 CRISPR-Cassystem. In some embodiments, the Type V CRISPR-Cas system is a V-B2CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system isa V-C CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cassystem is a V-D CRISPR-Cas system. In some embodiments, the Type VCRISPR-Cas system is a V-E CRISPR-Cas system. In some embodiments, theType V CRISPR-Cas system is a V-F1 CRISPR-Cas system. In someembodiments, the Type V CRISPR-Cas system is a V-F1 (V-U3) CRISPR-Cassystem. In some embodiments, the Type V CRISPR-Cas system is a V-F2CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system isa V-F3 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cassystem is a V-G CRISPR-Cas system. In some embodiments, the Type VCRISPR-Cas system is a V-H CRISPR-Cas system. In some embodiments, theType V CRISPR-Cas system is a V-I CRISPR-Cas system. In someembodiments, the Type V CRISPR-Cas system is a V-K (V-U5) CRISPR-Cassystem. In some embodiments, the Type V CRISPR-Cas system is a V-U1CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system isa V-U2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cassystem is a V-U4 CRISPR-Cas system. In some embodiments, the Type VCRISPR-Cas system includes a Cas12a (Cpf1), Cas12b (C2c1), Cas12c(C2c3), CasX, and/or Cas14.

In some embodiments the Class 2 system is a Type VI system. In someembodiments, the Type VI CRISPR-Cas system is a VI-A CRISPR-Cas system.In some embodiments, the Type VI CRISPR-Cas system is a VI-B1 CRISPR-Cassystem. In some embodiments, the Type VI CRISPR-Cas system is a VI-B2CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system isa VI-C CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cassystem is a VI-D CRISPR-Cas system. In some embodiments, the Type VICRISPR-Cas system includes a Cas13a (C2c2), Cas13b (Group 29/30),Cas13c, and/or Cas13d.

Specialized Cas-based Systems

In some embodiments, the system is a Cas-based system that is capable ofperforming a specialized function or activity. For example, the Casprotein may be fused, operably coupled to, or otherwise associated withone or more functionals domains. In certain example embodiments, the Casprotein may be a catalytically dead Cas protein (“dCas”) and/or havenickase activity. A nickase is a Cas protein that cuts only one strandof a double stranded target. In such embodiments, the dCas or nickaseprovide a sequence specific targeting functionality that delivers thefunctional domain to or proximate a target sequence. Example functionaldomains that may be fused to, operably coupled to, or otherwiseassociated with a Cas protein can be or include, but are not limited toa nuclear localization signal (NLS) domain, a nuclear export signal(NES) domain, a translational activation domain, a transcriptionalactivation domain (e.g. VP64, p65, MyoD1, HSF1, RTA, and SET7/9), atranslation initiation domain, a transcriptional repression domain(e.g., a KRAB domain, NuE domain, NcoR domain, and a SID domain such asa SID4X domain), a nuclease domain (e.g., FokI), a histone modificationdomain (e.g., a histone acetyltransferase), a lightinducible/controllable domain, a chemically inducible/controllabledomain, a transposase domain, a homologous recombination machinerydomain, a recombinase domain, an integrase domain, and combinationsthereof. Methods for generating catalytically dead Cas9 or a nickaseCas9 (WO 2014/204725, Ran et al. Cell. 2013 Sept 12; 154(6):1380-1389),Cas12 (Liu et al. Nature Communications, 8, 2095 (2017), and Cas13 (WO2019/005884, WO2019/060746) are known in the art and incorporated hereinby reference.

In some embodiments, the functional domains can have one or more of thefollowing activities: methylase activity, demethylase activity,translation activation activity, translation initiation activity,translation repression activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity, molecular switch activity, chemical inducibility,light inducibility, and nucleic acid binding activity. In someembodiments, the one or more functional domains may comprise epitopetags or reporters. Non-limiting examples of epitope tags includehistidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA)tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples ofreporters include, but are not limited to, glutathione-S-transferase(GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase(CAT) beta-galactosidase, beta-glucuronidase, luciferase, greenfluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP),yellow fluorescent protein (YFP), and auto-fluorescent proteinsincluding blue fluorescent protein (BFP).

The one or more functional domain(s) may be positioned at, near, and/orin proximity to a terminus of the effector protein (e.g., a Casprotein). In embodiments having two or more functional domains, each ofthe two can be positioned at or near or in proximity to a terminus ofthe effector protein (e.g., a Cas protein). In some embodiments, such asthose where the functional domain is operably coupled to the effectorprotein, the one or more functional domains can be tethered or linkedvia a suitable linker (including, but not limited to, GlySer linkers) tothe effector protein (e.g., a Cas protein). When there is more than onefunctional domain, the functional domains can be same or different. Insome embodiments, all the functional domains are the same. In someembodiments, all of the functional domains are different from eachother. In some embodiments, at least two of the functional domains aredifferent from each other. In some embodiments, at least two of thefunctional domains are the same as each other.

Other suitable functional domains can be found, for example, inInternational Application Publication No. WO 2019/018423.

Split CRISPR-Cas Systems

In some embodiments, the CRISPR-Cas system is a split CRISPR-Cas system.See e.g., Zetche et al., 2015. Nat. Biotechnol. 33(2): 139-142 and WO2019/018423, the compositions and techniques of which can be used inand/or adapted for use with the present invention. Split CRISPR-Casproteins are set forth herein and in documents incorporated herein byreference in further detail herein. In certain embodiments, each part ofa split CRISPR protein are attached to a member of a specific bindingpair, and when bound with each other, the members of the specificbinding pair maintain the parts of the CRISPR protein in proximity. Incertain embodiments, each part of a split CRISPR protein is associatedwith an inducible binding pair. An inducible binding pair is one whichis capable of being switched “on” or “off” by a protein or smallmolecule that binds to both members of the inducible binding pair. Insome embodiments, CRISPR proteins may preferably split between domains,leaving domains intact. In particular embodiments, said Cas splitdomains (e.g., RuvC and HNH domains in the case of Cas9) can besimultaneously or sequentially introduced into the cell such that saidsplit Cas domain(s) process the target nucleic acid sequence in thealgae cell. The reduced size of the split Cas compared to the wild typeCas allows other methods of delivery of the systems to the cells, suchas the use of cell penetrating peptides as described herein.

DNA and RNA Base Editing

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a base editing system. In someembodiments, a Cas protein is connected or fused to a nucleotidedeaminase. Thus, in some embodiments the Cas-based system can be a baseediting system. As used herein “base editing” refers generally to theprocess of polynucleotide modification via a CRISPR-Cas-based orCas-based system that does not include excising nucleotides to make themodification. Base editing can convert base pairs at precise locationswithout generating excess undesired editing byproducts that can be madeusing traditional CRISPR-Cas systems.

In certain example embodiments, the nucleotide deaminase may be a DNAbase editor used in combination with a DNA binding Cas protein such as,but not limited to, Class 2 Type II and Type V systems. Two classes ofDNA base editors are generally known: cytosine base editors (CBEs) andadenine base editors (ABEs). CBEs convert a C•G base pair into a T•Abase pair (Komor et al. 2016. Nature. 533:420-424; Nishida et al. 2016.Science. 353; and Li et al. Nat. Biotech. 36:324-327) and ABEs convertan A•T base pair to a G•C base pair. Collectively, CBEs and ABEs canmediate all four possible transition mutations (C to T, A to G, T to C,and G to A). Rees and Liu. 2018.Nat. Rev. Genet. 19(12): 770-788,particularly at FIGS. 1 b, 2 a-2 c, 3 a-3 f , and Table 1. In someembodiments, the base editing system includes a CBE and/or an ABE. Insome embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a base editing system. Rees andLiu. 2018. Nat. Rev. Gent. 19(12):770-788. Base editors also generallydo not need a DNA donor template and/or rely on homology-directedrepair. Komor et al. 2016. Nature. 533:420-424; Nishida et al. 2016.Science. 353; and Gaudeli et al. 2017. Nature. 551:464-471. Upon bindingto a target locus in the DNA, base pairing between the guide RNA of thesystem and the target DNA strand leads to displacement of a smallsegment of ssDNA in an “R-loop”. Nishimasu et al. Cell. 156:935-949. DNAbases within the ssDNA bubble are modified by the enzyme component, suchas a deaminase. In some systems, the catalytically disabled Cas proteincan be a variant or modified Cas can have nickase functionality and cangenerate a nick in the non-edited DNA strand to induce cells to repairthe non-edited strand using the edited strand as a template. Komor etal. 2016. Nature. 533:420-424; Nishida et al. 2016. Science. 353; andGaudeli et al. 2017. Nature. 551:464-471. Base editors may be furtherengineered to optimize conversion of nucleotides (e.g., A:T to G:C).Richter et al. 2020. Nature Biotechnology.doi.org/10.1038/s41587-020-0453-z.

Other Example Type V base editing systems are described in WO2018/213708, WO 2018/213726, PCT/US2018/067207, PCT/US2018/067225, andPCT/US2018/067307 which are incorporated by referenced herein.

In certain example embodiments, the base editing system may be a RNAbase editing system. As with DNA base editors, a nucleotide deaminasecapable of converting nucleotide bases may be fused to a Cas protein.However, in these embodiments, the Cas protein will need to be capableof binding RNA. Example RNA binding Cas proteins include, but are notlimited to, RNA-binding Cas9s such as Francisella novicida Cas9(“FnCas9”), and Class 2 Type VI Cas systems. The nucleotide deaminasemay be a cytidine deaminase or an adenosine deaminase, or an adenosinedeaminase engineered to have cytidine deaminase activity. In certainexample embodiments, the RNA based editor may be used to delete orintroduce a post-translation modification site in the expressed mRNA. Incontrast to DNA base editors, whose edits are permanent in the modifiedcell, RNA base editors can provide edits where finer temporal controlmay be needed, for example in modulating a particular immune response.Example Type VI RNA-base editing systems are described in Cox et al.2017. Science 358: 1019-1027, WO 2019/005884, WO 2019/005886, WO2019/071048, PCT/US20018/05179, PCT/US2018/067207, which areincorporated herein by reference. An example FnCas9 system that may beadapted for RNA base editing purposes is described in WO 2016/106236,which is incorporated herein by reference.

An example method for delivery of base-editing systems, including use ofa split-intein approach to divide CBE and ABE into reconstituble halves,is described in Levy et al. Nature Biomedical Engineeringdoi.org/10.1038/s41441-019-0505-5 (2019), which is incorporated hereinby reference.

Prime Editors

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a prime editing system (See e.g.,Anzalone et al. 2019. Nature. 576: 149-157). Like base editing systems,prime editing systems can be capable of targeted modification of apolynucleotide without generating double stranded breaks and does notrequire donor templates. Further prime editing systems can be capable ofall 12 possible combination swaps. Prime editing can operate via a“search-and-replace” methodology and can mediate targeted insertions,deletions, all 12 possible base-to-base conversion, and combinationsthereof. Generally, a prime editing system, as exemplified by PE1, PE2,and PE3 (Id.), can include a reverse transcriptase fused or otherwisecoupled or associated with an RNA-programmable nickase, and aprime-editing extended guide RNA (pegRNA) to facility direct copying ofgenetic information from the extension on the pegRNA into the targetpolynucleotide. Embodiments that can be used with the present inventioninclude these and variants thereof. Prime editing can have the advantageof lower off-target activity than traditional CRIPSR-Cas systems alongwith few byproducts and greater or similar efficiency as compared totraditional CRISPR-Cas systems.

In some embodiments, the prime editing guide molecule can specify boththe target polynucleotide information (e.g., sequence) and contain a newpolynucleotide cargo that replaces target polynucleotides. To initiatetransfer from the guide molecule to the target polynucleotide, the PEsystem can nick the target polynucleotide at a target side to expose a3′hydroxyl group, which can prime reverse transcription of anedit-encoding extension region of the guide molecule (e.g., a primeediting guide molecule or peg guide molecule) directly into the targetsite in the target polynucleotide. See e.g., Anzalone et al. 2019.Nature. 576: 149-157, particularly at FIGS. 1 b, 1 c , relateddiscussion, and Supplementary discussion.

In some embodiments, a prime editing system can be composed of a Caspolypeptide having nickase activity, a reverse transcriptase, and aguide molecule. The Cas polypeptide can lack nuclease activity. Theguide molecule can include a target binding sequence as well as a primerbinding sequence and a template containing the edited polynucleotidesequence. The guide molecule, Cas polypeptide, and/or reversetranscriptase can be coupled together or otherwise associate with eachother to form an effector complex and edit a target sequence. In someembodiments, the Cas polypeptide is a Class 2, Type V Cas polypeptide.In some embodiments, the Cas polypeptide is a Cas9 polypeptide (e.g., isa Cas9 nickase). In some embodiments, the Cas polypeptide is fused tothe reverse transcriptase. In some embodiments, the Cas polypeptide islinked to the reverse transcriptase.

In some embodiments, the prime editing system can be a PE1 system orvariant thereof, a PE2 system or variant thereof, or a PE3 (e.g., PE3,PE3b) system. See e.g., Anzalone et al. 2019. Nature. 576: 149-157,particularly at pgs. 2-3, FIGS. 2 a, 3 a-3 f, 4 a-4 b , Extended dataFIGS. 3A-3B, 4 ,

The peg guide molecule can be about 10 to about 200 or more nucleotidesin length, such as 10 to/or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, or 200 or more nucleotides in length.Optimization of the peg guide molecule can be accomplished as describedin Anzalone et al. 2019. Nature. 576: 149-157, particularly at pg. 3,FIGS. 2A-2B, and Extended Data FIGS. 5A-D.

CRISPR Associated Transposase (CAST) Systems

In some embodiments, a polynucleotide of the present invention describedelsewhere herein can be modified using a CRISPR Associated Transposase(“CAST”) system. CAST system can include a Cas protein that iscatalytically inactive, or engineered to be catalytically active, andfurther comprises a transposase (or subunits thereof) that catalyzeRNA-guided DNA transposition. Such systems are able to insert DNAsequences at a target site in a DNA molecule without relying on hostcell repair machinery. CAST systems can be Class1 or Class 2 CASTsystems. An example Class 1 system is described in Klompe et al. Nature,doi:10.1038/s41586-019-1323, which is in incorporated herein byreference. An example Class 2 system is described in Strecker et al.Science. 10/1126/science. aax9181 (2019), and PCT/US2019/066835 whichare incorporated herein by reference.

Guide Molecules

The CRISPR-Cas or Cas-Based system described herein can, in someembodiments, include one or more guide molecules. The terms guidemolecule, guide sequence and guide polynucleotide, refer topolynucleotides capable of guiding Cas to a target genomic locus and areused interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence.The guide molecule can be a polynucleotide.

The ability of a guide sequence (within a nucleic acid-targeting guideRNA) to direct sequence-specific binding of a nucleic acid-targetingcomplex to a target nucleic acid sequence may be assessed by anysuitable assay. For example, the components of a nucleic acid-targetingCRISPR system sufficient to form a nucleic acid-targeting complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target nucleic acid sequence, such as bytransfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay (Qui et al. 2004. BioTechniques. 36(4)702-707).Similarly, cleavage of a target nucleic acid sequence may be evaluatedin a test tube by providing the target nucleic acid sequence, componentsof a nucleic acid-targeting complex, including the guide sequence to betested and a control guide sequence different from the test guidesequence, and comparing binding or rate of cleavage at the targetsequence between the test and control guide sequence reactions. Otherassays are possible and will occur to those skilled in the art.

In some embodiments, the guide molecule is an RNA. The guide molecule(s)(also referred to interchangeably herein as guide polynucleotide andguide sequence) that are included in the CRISPR-Cas or Cas based systemcan be any polynucleotide sequence having sufficient complementaritywith a target nucleic acid sequence to hybridize with the target nucleicacid sequence and direct sequence-specific binding of a nucleicacid-targeting complex to the target nucleic acid sequence. In someembodiments, the degree of complementarity, when optimally aligned usinga suitable alignment algorithm, can be about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences, non-limiting examples of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

A guide sequence, and hence a nucleic acid-targeting guide may beselected to target any target nucleic acid sequence. The target sequencemay be DNA. The target sequence may be any RNA sequence. In someembodiments, the target sequence may be a sequence within an RNAmolecule selected from the group consisting of messenger RNA (mRNA),pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA),small interfering RNA (siRNA), small nuclear RNA (snRNA), smallnucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA(ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA(scRNA). In some preferred embodiments, the target sequence may be asequence within an RNA molecule selected from the group consisting ofmRNA, pre-mRNA, and rRNA. In some preferred embodiments, the targetsequence may be a sequence within an RNA molecule selected from thegroup consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide is selected toreduce the degree secondary structure within the nucleic acid-targetingguide. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide participate in self-complementary base pairing whenoptimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr andGM Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19,or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27nt, from 27 to 30 nt, e.g., 27, 28, 29, or 30 nt, from 30 to 35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

The “tracrRNA” sequence or analogous terms includes any polynucleotidesequence that has sufficient complementarity with a crRNA sequence tohybridize. In some embodiments, the degree of complementarity betweenthe tracrRNA sequence and crRNA sequence along the length of the shorterof the two when optimally aligned is about or more than about 25%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In someembodiments, the tracr sequence is about or more than about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or morenucleotides in length. In some embodiments, the tracr sequence and crRNAsequence are contained within a single transcript, such thathybridization between the two produces a transcript having a secondarystructure, such as a hairpin.

In general, degree of complementarity is with reference to the optimalalignment of the sca sequence and tracr sequence, along the length ofthe shorter of the two sequences. Optimal alignment may be determined byany suitable alignment algorithm, and may further account for secondarystructures, such as self-complementarity within either the sca sequenceor tracr sequence. In some embodiments, the degree of complementaritybetween the tracr sequence and sca sequence along the length of theshorter of the two when optimally aligned is about or more than about25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.

In some embodiments, the degree of complementarity between a guidesequence and its corresponding target sequence can be about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; a guide orRNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, 75, or more nucleotides in length; or guide or RNA or sgRNA can beless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length; and tracr RNA can be 30 or 50 nucleotides inlength. In some embodiments, the degree of complementarity between aguide sequence and its corresponding target sequence is greater than94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or99% or 99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88%or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementaritybetween the sequence and the guide, with it advantageous that off targetis 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97%or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between thesequence and the guide.

In some embodiments according to the invention, the guide RNA (capableof guiding Cas to a target locus) may comprise (1) a guide sequencecapable of hybridizing to a genomic target locus in the eukaryotic cell;(2) a tracr sequence; and (3) a tracr mate sequence. All (1) to (3) mayreside in a single RNA, i.e., an sgRNA (arranged in a 5′ to 3′orientation), or the tracr RNA may be a different RNA than the RNAcontaining the guide and tracr sequence. The tracr hybridizes to thetracr mate sequence and directs the CRISPR/Cas complex to the targetsequence. Where the tracr RNA is on a different RNA than the RNAcontaining the guide and tracr sequence, the length of each RNA may beoptimized to be shortened from their respective native lengths, and eachmay be independently chemically modified to protect from degradation bycellular RNase or otherwise increase stability.

Many modifications to guide sequences are known in the art and arefurther contemplated within the context of this invention. Variousmodifications may be used to increase the specificity of binding to thetarget sequence and/or increase the activity of the Cas protein and/orreduce off-target effects. Example guide sequence modifications aredescribed in PCT US2019/045582, specifically paragraphs -[0333]. whichis incorporated herein by reference.

Target Sequences, PAMs, and PFSs Target Sequences

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. The term “target RNA” refersto an RNA polynucleotide being or comprising the target sequence. Inother words, the target polynucleotide can be a polynucleotide or a partof a polynucleotide to which a part of the guide sequence is designed tohave complementarity with and to which the effector function mediated bythe complex comprising the CRISPR effector protein and a guide moleculeis to be directed. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell.

The guide sequence can specifically bind a target sequence in a targetpolynucleotide. The target polynucleotide may be DNA. The targetpolynucleotide may be RNA. The target polynucleotide can have one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. or more) targetsequences. The target polynucleotide can be on a vector. The targetpolynucleotide can be genomic DNA. The target polynucleotide can beepisomal. Other forms of the target polynucleotide are describedelsewhere herein.

The target sequence may be DNA. The target sequence may be any RNAsequence. In some embodiments, the target sequence may be a sequencewithin an RNA molecule selected from the group consisting of messengerRNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA),micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA(snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA),non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and smallcytoplasmatic RNA (scRNA). In some preferred embodiments, the targetsequence (also referred to herein as a target polynucleotide) may be asequence within an RNA molecule selected from the group consisting ofmRNA, pre-mRNA, and rRNA. In some preferred embodiments, the targetsequence may be a sequence within an RNA molecule selected from thegroup consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

PAM and PFS Elements

PAM elements are sequences that can be recognized and bound by Casproteins. Cas proteins/effector complexes can then unwind the dsDNA at aposition adjacent to the PAM element. It will be appreciated that Casproteins and systems that include them that target RNA do not requirePAM sequences (Marraffini et al. 2010. Nature. 463:568-571). Instead,many rely on PFSs, which are discussed elsewhere herein. In certainembodiments, the target sequence should be associated with a PAM(protospacer adjacent motif) or PFS (protospacer flanking sequence orsite), that is, a short sequence recognized by the CRISPR complex.Depending on the nature of the CRISPR-Cas protein, the target sequenceshould be selected, such that its complementary sequence in the DNAduplex (also referred to herein as the non-target sequence) is upstreamor downstream of the PAM. In the embodiments, the complementary sequenceof the target sequence is downstream or 3′ of the PAM or upstream or 5′of the PAM. The precise sequence and length requirements for the PAMdiffer depending on the Cas protein used, but PAMs are typically 2-5base pair sequences adjacent the protospacer (that is, the targetsequence). Examples of the natural PAM sequences for different Casproteins are provided herein below and the skilled person will be ableto identify further PAM sequences for use with a given Cas protein.

The ability to recognize different PAM sequences depends on the Caspolypeptide(s) included in the system. See e.g., Gleditzsch et al. 2019.RNA Biology. 16(4):504-517. Table 3 below shows several Cas polypeptidesand the PAM sequence they recognize.

TABLE 3 Example PAM Sequences Cas Protein PAM Sequence SpCas9 NGG/NRGSaCas9 NGRRT or NGRRN NmeCas9 NNNNGATT CjCas9 NNNNRYAC StCas9 NNAGAAWCas12a (Cpf1) (including LbCpf1 and AsCpf1) TTTV Cas12b (C2c1) TTT, TTA,and TTC Cas12c (C2c3) TA Cas12d (CasY) TA Cas12e (CasX) 5′-TTCN-3′

In a preferred embodiment, the CRISPR effector protein may recognize a3′ PAM. In certain embodiments, the CRISPR effector protein mayrecognize a 3′ PAM which is 5′H, wherein H is A, C or U.

Further, engineering of the PAM Interacting (PI) domain on the Casprotein may allow programing of PAM specificity, improve target siterecognition fidelity, and increase the versatility of the CRISPR-Casprotein, for example as described for Cas9 in Kleinstiver BP et al.Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature.2015 Jul 23;523(7561):481-5. doi: 10.1038/nature14592. As furtherdetailed herein, the skilled person will understand that Cas 13 proteinsmay be modified analogously. Gao et al, “Engineered Cpf1 Enzymes withAltered PAM Specificities,” bioRxiv 091611; doi:http://dx.doi.org/10.1101/091611 (Dec. 4, 2016). Doench et al. created apool of sgRNAs, tiling across all possible target sites of a panel ofsix endogenous mouse and three endogenous human genes and quantitativelyassessed their ability to produce null alleles of their target gene byantibody staining and flow cytometry. The authors showed thatoptimization of the PAM improved activity and also provided an on-linetool for designing sgRNAs.

PAM sequences can be identified in a polynucleotide using an appropriatedesign tool, which are commercially available as well as online. Suchfreely available tools include, but are not limited to, CRISPRFinder andCRISPRTarget. Mojica et al. 2009. Microbiol. 155(Pt. 3):733-740; Atschulet al. 1990. J. Mol. Biol. 215:403-410; Biswass et al. 2013 RNA Biol.10:817-827; and Grissa et al. 2007. Nucleic Acid Res. 35:W52-57.Experimental approaches to PAM identification can include, but are notlimited to, plasmid depletion assays (Jiang et al. 2013. Nat.Biotechnol. 31:233-239; Esvelt et al. 2013. Nat. Methods. 10:1116-1121;Kleinstiver et al. 2015. Nature. 523:481-485), screened by ahigh-throughput in vivo model called PAM-SCNAR (Pattanayak et al. 2013.Nat. Biotechnol. 31:839-843 and Leenay et al. 2016.Mol. Cell. 16:253),and negative screening (Zetsche et al. 2015. Cell. 163:759-771).

As previously mentioned, CRISPR-Cas systems that target RNA do nottypically rely on PAM sequences. Instead, such systems typicallyrecognize protospacer flanking sites (PFSs) instead of PAMs Thus, TypeVI CRISPR-Cas systems typically recognize protospacer flanking sites(PFSs) instead of PAMs. PFSs represents an analogue to PAMs for RNAtargets. Type VI CRISPR-Cas systems employ a Cas13. Some Cas13 proteinsanalyzed to date, such as Cas13a (C2c2) identified from Leptotrichiashahii (LShCAs13a) have a specific discrimination against G at the 3′end of the target RNA. The presence of a C at the corresponding crRNArepeat site can indicate that nucleotide pairing at this position isrejected. However, some Cas13 proteins (e.g., LwaCAs13a and PspCas13b)do not seem to have a PFS preference. See e.g., Gleditzsch et al. 2019.RNA Biology. 16(4):504-517.

Some Type VI proteins, such as subtype B, have 5′-recognition of D (G,T, A) and a 3′-motif requirement of NAN or NNA. One example is theCas13b protein identified in Bergeyella zoohelcum (BzCas13b). See e.g.,Gleditzsch et al. 2019. RNA Biology. 16(4):504-517.

Overall Type VI CRISPR-Cas systems appear to have less restrictive rulesfor substrate (e.g., target sequence) recognition than those that targetDNA (e.g., Type V and type II).

Zinc Finger Nucleases

In some embodiments, the polynucleotide is modified using a Zinc Fingernuclease or system thereof. One type of programmable DNA-binding domainis provided by artificial zinc-finger (ZF) technology, which involvesarrays of ZF modules to target new DNA-binding sites in the genome. Eachfinger module in a ZF array targets three DNA bases. A customized arrayof individual zinc finger domains is assembled into a ZF protein (ZFP).

ZFPs can comprise a functional domain. The first synthetic zinc fingernucleases (ZFNs) were developed by fusing a ZF protein to the catalyticdomain of the Type IIS restriction enzyme FokI. (Kim, Y. G. et al.,1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S.A.91, 883-887; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zincfinger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S.A.93, 1156-1160). Increased cleavage specificity can be attained withdecreased off target activity by use of paired ZFN heterodimers, eachtargeting different nucleotide sequences separated by a short spacer.(Doyon, Y. et al., 2011, Enhancing zinc-finger-nuclease activity withimproved obligate heterodimeric architectures. Nat. Methods 8, 74-79).ZFPs can also be designed as transcription activators and repressors andhave been used to target many genes in a wide variety of organisms.Exemplary methods of genome editing using ZFNs can be found for examplein U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136, 6,824,978,6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215, 7,220,719,7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and 6,479,626,all of which are specifically incorporated by reference.

TALE Nucleases

In some embodiments, a TALE nuclease or TALE nuclease system can be usedto modify a polynucleotide. In some embodiments, the methods providedherein use isolated, non-naturally occurring, recombinant or engineeredDNA binding proteins that comprise TALE monomers or TALE monomers orhalf monomers as a part of their organizational structure that enablethe targeting of nucleic acid sequences with improved efficiency andexpanded specificity.

Naturally occurring TALEs or “wild type TALEs” are nucleic acid bindingproteins secreted by numerous species of proteobacteria. TALEpolypeptides contain a nucleic acid binding domain composed of tandemrepeats of highly conserved monomer polypeptides that are predominantly33, 34 or 35 amino acids in length and that differ from each othermainly in amino acid positions 12 and 13. In advantageous embodimentsthe nucleic acid is DNA. As used herein, the term “polypeptidemonomers”, “TALE monomers” or “monomers” will be used to refer to thehighly conserved repetitive polypeptide sequences within the TALEnucleic acid binding domain and the term “repeat variable di-residues”or “RVD” will be used to refer to the highly variable amino acids atpositions 12 and 13 of the polypeptide monomers. As provided throughoutthe disclosure, the amino acid residues of the RVD are depicted usingthe IUPAC single letter code for amino acids. A general representationof a TALE monomer which is comprised within the DNA binding domain isX₁₋₁₁-(X₁₂X₁₃)-X₁₄₋₃₃ or 34 or 35, where the subscript indicates theamino acid position and X represents any amino acid. X₁₂X₁₃ indicate theRVDs. In some polypeptide monomers, the variable amino acid at position13 is missing or absent and in such monomers, the RVD consists of asingle amino acid. In such cases the RVD may be alternativelyrepresented as X*, where X represents X₁₂ and (*) indicates that X₁₃ isabsent. The DNA binding domain comprises several repeats of TALEmonomers and this may be represented as (X₁₋₁₁-(X₁₂X₁₃)-X₁₄₋₃₃ or 34 or35)_(z), where in an advantageous embodiment, z is at least 5 to 40. Ina further advantageous embodiment, z is at least 10 to 26.

The TALE monomers can have a nucleotide binding affinity that isdetermined by the identity of the amino acids in its RVD. For example,polypeptide monomers with an RVD of NI can preferentially bind toadenine (A), monomers with an RVD of NG can preferentially bind tothymine (T), monomers with an RVD of HD can preferentially bind tocytosine (C) and monomers with an RVD of NN can preferentially bind toboth adenine (A) and guanine (G). In some embodiments, monomers with anRVD of IG can preferentially bind to T. Thus, the number and order ofthe polypeptide monomer repeats in the nucleic acid binding domain of aTALE determines its nucleic acid target specificity. In someembodiments, monomers with an RVD of NS can recognize all four basepairs and can bind to A, T, G or C. The structure and function of TALEsis further described in, for example, Moscou et al., Science 326:1501(2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al.,Nature Biotechnology 29:149-153 (2011).

The polypeptides used in methods of the invention can be isolated,non-naturally occurring, recombinant or engineered nucleic acid-bindingproteins that have nucleic acid or DNA binding regions containingpolypeptide monomer repeats that are designed to target specific nucleicacid sequences.

As described herein, polypeptide monomers having an RVD of HN or NHpreferentially bind to guanine and thereby allow the generation of TALEpolypeptides with high binding specificity for guanine containing targetnucleic acid sequences. In some embodiments, polypeptide monomers havingRVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS canpreferentially bind to guanine. In some embodiments, polypeptidemonomers having RVDs RN, NK, NQ, HH, KH, RH, SS and SN canpreferentially bind to guanine and can thus allow the generation of TALEpolypeptides with high binding specificity for guanine containing targetnucleic acid sequences. In some embodiments, polypeptide monomers havingRVDs HH, KH, NH, NK, NQ, RH, RN and SS can preferentially bind toguanine and thereby allow the generation of TALE polypeptides with highbinding specificity for guanine containing target nucleic acidsequences. In some embodiments, the RVDs that have high bindingspecificity for guanine are RN, NH RH and KH. Furthermore, polypeptidemonomers having an RVD of NV can preferentially bind to adenine andguanine. In some embodiments, monomers having RVDs of H*, HA, KA, N*,NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and thyminewith comparable affinity.

The predetermined N-terminal to C-terminal order of the one or morepolypeptide monomers of the nucleic acid or DNA binding domaindetermines the corresponding predetermined target nucleic acid sequenceto which the polypeptides of the invention will bind. As used herein themonomers and at least one or more half monomers are “specificallyordered to target” the genomic locus or gene of interest. In plantgenomes, the natural TALE-binding sites always begin with a thymine (T),which may be specified by a cryptic signal within the non-repetitiveN-terminus of the TALE polypeptide; in some cases, this region may bereferred to as repeat 0. In animal genomes, TALE binding sites do notnecessarily have to begin with a thymine (T) and polypeptides of theinvention may target DNA sequences that begin with T, A, G or C. Thetandem repeat of TALE monomers always ends with a half-length repeat ora stretch of sequence that may share identity with only the first 20amino acids of a repetitive full-length TALE monomer and this halfrepeat may be referred to as a half-monomer. Therefore, it follows thatthe length of the nucleic acid or DNA being targeted is equal to thenumber of full monomers plus two.

As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),TALE polypeptide binding efficiency may be increased by including aminoacid sequences from the “capping regions” that are directly N-terminalor C-terminal of the DNA binding region of naturally occurring TALEsinto the engineered TALEs at positions N-terminal or C-terminal of theengineered TALE DNA binding region. Thus, in certain embodiments, theTALE polypeptides described herein further comprise an N-terminalcapping region and/or a C-terminal capping region.

An exemplary amino acid sequence of a N-terminal capping region is:

M D P I R S R T P S P A R E L L S G P Q P D G V Q P T A D R G V S P P A GG P L D G L P A R R T M S R T R L P S P P A P S P A F S A D S F S D L L R Q F D P S LF N T S L F D S L P P F G A H H T E A A T G E W D E V Q S G L R A A D A P P P T MR V A V T A A R P P R A K P A P R R R A A Q P S D A S P A A Q V D L R T L G Y S QQ Q Q E K I K P K V R S T V A Q H H E A L V G H G F T H A H I V A L S Q H P A A LG T V A V K Y Q D M I A A L P E A T H E A I V G V G K Q W S G A R A L E A L L T VA G E L R G P P L Q L D T G Q L L K I A K R G G V T A V E A V H A W R N A L T G AP L N (SEQ ID NO: 3)

An exemplary amino acid sequence of a C-terminal capping region is:

R P A L E S I V A Q L S R P D P A L A A L T N D H L V A L A C L G G R P AL D A V K K G L P H A P A L I K R T N R R I P E R T S H R V A D H A Q V V R V L GF F Q C H S H P A Q A F D D A M T Q F G M S R H G L L Q L F R R V G V T E L E A RS G T L P P A S Q R W D R I L Q A S G M K R A K P S P T S T Q T P D Q A S L H A F AD S L E R D L D A P S P M H E G D Q T R A S (SEQ ID NO: 4)

As used herein the predetermined “N-terminus” to “C terminus”orientation of the N-terminal capping region, the DNA binding domaincomprising the repeat TALE monomers and the C-terminal capping regionprovide structural basis for the organization of different domains inthe d-TALEs or polypeptides of the invention.

The entire N-terminal and/or C-terminal capping regions are notnecessary to enhance the binding activity of the DNA binding region.Therefore, in certain embodiments, fragments of the N-terminal and/orC-terminal capping regions are included in the TALE polypeptidesdescribed herein.

In certain embodiments, the TALE polypeptides described herein contain aN-terminal capping region fragment that included at least 10, 20, 30,40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140,147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270amino acids of an N-terminal capping region. In certain embodiments, theN-terminal capping region fragment amino acids are of the C-terminus(the DNA-binding region proximal end) of an N-terminal capping region.As described in Zhang et al., Nature Biotechnology 29:149-153 (2011),N-terminal capping region fragments that include the C-terminal 240amino acids enhance binding activity equal to the full length cappingregion, while fragments that include the C-terminal 147 amino acidsretain greater than 80% of the efficacy of the full length cappingregion, and fragments that include the C-terminal 117 amino acids retaingreater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain aC-terminal capping region fragment that included at least 6, 10, 20, 30,37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155,160, 170, 180 amino acids of a C-terminal capping region. In certainembodiments, the C-terminal capping region fragment amino acids are ofthe N-terminus (the DNA-binding region proximal end) of a C-terminalcapping region. As described in Zhang et al., Nature Biotechnology29:149-153 (2011), C-terminal capping region fragments that include theC-terminal 68 amino acids enhance binding activity equal to thefull-length capping region, while fragments that include the C-terminal20 amino acids retain greater than 50% of the efficacy of thefull-length capping region.

In certain embodiments, the capping regions of the TALE polypeptidesdescribed herein do not need to have identical sequences to the cappingregion sequences provided herein. Thus, in some embodiments, the cappingregion of the TALE polypeptides described herein have sequences that areat least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identical or share identity to the capping region aminoacid sequences provided herein. Sequence identity is related to sequencehomology. Homology comparisons may be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences. In some preferred embodiments, the capping region of the TALEpolypeptides described herein have sequences that are at least 95%identical or share identity to the capping region amino acid sequencesprovided herein.

Sequence homologies can be generated by any of a number of computerprograms known in the art, which include but are not limited to BLAST orFASTA. Suitable computer programs for carrying out alignments like theGCG Wisconsin Bestfit package may also be used. Once the software hasproduced an optimal alignment, it is possible to calculate % homology,preferably % sequence identity. The software typically does this as partof the sequence comparison and generates a numerical result.

In some embodiments described herein, the TALE polypeptides of theinvention include a nucleic acid binding domain linked to the one ormore effector domains. The terms “effector domain” or “regulatory andfunctional domain” refer to a polypeptide sequence that has an activityother than binding to the nucleic acid sequence recognized by thenucleic acid binding domain. By combining a nucleic acid binding domainwith one or more effector domains, the polypeptides of the invention maybe used to target the one or more functions or activities mediated bythe effector domain to a particular target DNA sequence to which thenucleic acid binding domain specifically binds.

In some embodiments of the TALE polypeptides described herein, theactivity mediated by the effector domain is a biological activity. Forexample, in some embodiments the effector domain is a transcriptionalinhibitor (i.e., a repressor domain), such as an mSin interaction domain(SID). SID4X domain or a Krüppel-associated box (KRAB) or fragments ofthe KRAB domain. In some embodiments the effector domain is an enhancerof transcription (i.e., an activation domain), such as the VP16, VP64 orp65 activation domain. In some embodiments, the nucleic acid binding islinked, for example, with an effector domain that includes but is notlimited to a transposase, integrase, recombinase, resolvase, invertase,protease, DNA methyltransferase, DNA demethylase, histone acetylase,histone deacetylase, nuclease, transcriptional repressor,transcriptional activator, transcription factor recruiting, proteinnuclear-localization signal or cellular uptake signal.

In some embodiments, the effector domain is a protein domain whichexhibits activities which include but are not limited to transposaseactivity, integrase activity, recombinase activity, resolvase activity,invertase activity, protease activity, DNA methyltransferase activity,DNA demethylase activity, histone acetylase activity, histonedeacetylase activity, nuclease activity, nuclear-localization signalingactivity, transcriptional repressor activity, transcriptional activatoractivity, transcription factor recruiting activity, or cellular uptakesignaling activity. Other preferred embodiments of the invention mayinclude any combination of the activities described herein.

Meganucleases

In some embodiments, a meganuclease or system thereof can be used tomodify a polynucleotide. Meganucleases, which are endodeoxyribonucleasescharacterized by a large recognition site (double-stranded DNA sequencesof 12 to 40 base pairs). Exemplary methods for using meganucleases canbe found in U.S. Pat. Nos. 8,163,514, 8,133,697, 8,021,867, 8,119,361,8,119,381, 8,124,369, and 8,129,134, which are specifically incorporatedby reference.

Sequences Related to Nucleus Targeting and Transportation

In some embodiments, one or more components (e.g., the Cas proteinand/or deaminase, Zn Finger protein, TALE, or meganuclease) in thecomposition for engineering cells may comprise one or more sequencesrelated to nucleus targeting and transportation. Such sequence mayfacilitate the one or more components in the composition for targeting asequence within a cell. In order to improve targeting of the CRISPR-Casprotein and/or the nucleotide deaminase protein or catalytic domainthereof used in the methods of the present disclosure to the nucleus, itmay be advantageous to provide one or both of these components with oneor more nuclear localization sequences (NLSs).

In some embodiments, the NLSs used in the context of the presentdisclosure are heterologous to the proteins. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 5)or PKKKRKVEAS (SEQ ID NO: 6); the NLS from nucleoplasmin (e.g., thenucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ IDNO: 7)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ IDNO: 8) or RQRRNELKRSP (SEQ ID NO: 9); the hRNPA1 M9 NLS having thesequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 10); thesequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 11) ofthe IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:12) and PPKKARED (SEQ ID NO: 13) of the myoma T protein; the sequencePQPKKKPL (SEQ ID NO: 14) of human p53; the sequence SALIKKKKKMAP (SEQ IDNO: 15) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 16) andPKQKKRK (SEQ ID NO: 17) of the influenza virus NS1; the sequenceRKLKKKIKKL (SEQ ID NO: 18) of the Hepatitis virus delta antigen; thesequence REKKKFLKRR (SEQ ID NO: 19) of the mouse Mx1 protein; thesequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 20) of the humanpoly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ IDNO: 21) of the steroid hormone receptors (human) glucocorticoid. Ingeneral, the one or more NLSs are of sufficient strength to driveaccumulation of the DNA-targeting Cas protein in a detectable amount inthe nucleus of a eukaryotic cell. In general, strength of nuclearlocalization activity may derive from the number of NLSs in theCRISPR-Cas protein, the particular NLS(s) used, or a combination ofthese factors. Detection of accumulation in the nucleus may be performedby any suitable technique. For example, a detectable marker may be fusedto the nucleic acid-targeting protein, such that location within a cellmay be visualized, such as in combination with a means for detecting thelocation of the nucleus (e.g., a stain specific for the nucleus such asDAPI). Cell nuclei may also be isolated from cells, the contents ofwhich may then be analyzed by any suitable process for detectingprotein, such as immunohistochemistry, Western blot, or enzyme activityassay. Accumulation in the nucleus may also be determined indirectly,such as by an assay for the effect of nucleic acid-targeting complexformation (e.g., assay for deaminase activity) at the target sequence,or assay for altered gene expression activity affected by DNA-targetingcomplex formation and/or DNA-targeting), as compared to a control notexposed to the CRISPR-Cas protein and deaminase protein, or exposed to aCRISPR-Cas and/or deaminase protein lacking the one or more NLSs.

The CRISPR-Cas and/or nucleotide deaminase proteins may be provided with1 or more, such as with, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moreheterologous NLSs. In some embodiments, the proteins comprises about ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or nearthe amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more NLSs at or near the carboxy-terminus, or a combination ofthese (e.g., zero or at least one or more NLS at the amino-terminus andzero or at one or more NLS at the carboxy terminus). When more than oneNLS is present, each may be selected independently of the others, suchthat a single NLS may be present in more than one copy and/or incombination with one or more other NLSs present in one or more copies.In some embodiments, an NLS is considered near the N- or C-terminus whenthe nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 40, 50, or more amino acids along the polypeptide chain fromthe N- or C-terminus. In preferred embodiments of the CRISPR-Casproteins, an NLS attached to the C-terminal of the protein.

In certain embodiments, the CRISPR-Cas protein and the deaminase proteinare delivered to the cell or expressed within the cell as separateproteins. In these embodiments, each of the CRISPR-Cas and deaminaseprotein can be provided with one or more NLSs as described herein. Incertain embodiments, the CRISPR-Cas and deaminase proteins are deliveredto the cell or expressed with the cell as a fusion protein. In theseembodiments one or both of the CRISPR-Cas and deaminase protein isprovided with one or more NLSs. Where the nucleotide deaminase is fusedto an adaptor protein (such as MS2) as described above, the one or moreNLS can be provided on the adaptor protein, provided that this does notinterfere with aptamer binding. In particular embodiments, the one ormore NLS sequences may also function as linker sequences between thenucleotide deaminase and the CRISPR-Cas protein.

In certain embodiments, guides of the disclosure comprise specificbinding sites (e.g. aptamers) for adapter proteins, which may be linkedto or fused to an nucleotide deaminase or catalytic domain thereof. Whensuch a guide forms a CRISPR complex (e.g., CRISPR-Cas protein binding toguide and target) the adapter proteins bind and, the nucleotidedeaminase or catalytic domain thereof associated with the adapterprotein is positioned in a spatial orientation which is advantageous forthe attributed function to be effective.

The skilled person will understand that modifications to the guide whichallow for binding of the adapter + nucleotide deaminase, but not properpositioning of the adapter + nucleotide deaminase (e.g., due to sterichindrance within the three dimensional structure of the CRISPR complex)are modifications which are not intended. The one or more modified guidemay be modified at the tetra loop, the stem loop 1, stem loop 2, or stemloop 3, as described herein, preferably at either the tetra loop or stemloop 2, and in some cases at both the tetra loop and stem loop 2.

In some embodiments, a component (e.g., the dead Cas protein, thenucleotide deaminase protein or catalytic domain thereof, or acombination thereof) in the systems may comprise one or more nuclearexport signals (NES), one or more nuclear localization signals (NLS), orany combinations thereof. In some cases, the NES may be an HIV Rev NES.In certain cases, the NES may be MAPK NES. When the component is aprotein, the NES or NLS may be at the C terminus of component.Alternatively, or additionally, the NES or NLS may be at the N terminusof component. In some examples, the Cas protein and optionally saidnucleotide deaminase protein or catalytic domain thereof comprise one ormore heterologous nuclear export signal(s) (NES(s)) or nuclearlocalization signal(s) (NLS(s)), preferably an HIV Rev NES or MAPK NES,preferably C-terminal.

Templates

In some embodiments, the composition for engineering cells comprise atemplate, e.g., a recombination template. A template may be a componentof another vector as described herein, contained in a separate vector,or provided as a separate polynucleotide. In some embodiments, arecombination template is designed to serve as a template in homologousrecombination, such as within or near a target sequence nicked orcleaved by a nucleic acid-targeting effector protein as a part of anucleic acid-targeting complex.

In an embodiment, the template nucleic acid alters the sequence of thetarget position. In an embodiment, the template nucleic acid results inthe incorporation of a modified, or non-naturally occurring base intothe target nucleic acid.

The template sequence may undergo a breakage mediated or catalyzedrecombination with the target sequence. In an embodiment, the templatenucleic acid may include sequence that corresponds to a site on thetarget sequence that is cleaved by a Cas protein mediated cleavageevent. In an embodiment, the template nucleic acid may include sequencethat corresponds to both, a first site on the target sequence that iscleaved in a first Cas protein mediated event, and a second site on thetarget sequence that is cleaved in a second Cas protein mediated event.

In certain embodiments, the template nucleic acid can include sequencewhich results in an alteration in the coding sequence of a translatedsequence, e.g., one which results in the substitution of one amino acidfor another in a protein product, e.g., transforming a mutant alleleinto a wild type allele, transforming a wild type allele into a mutantallele, and/or introducing a stop codon, insertion of an amino acidresidue, deletion of an amino acid residue, or a nonsense mutation. Incertain embodiments, the template nucleic acid can include sequencewhich results in an alteration in a non-coding sequence, e.g., analteration in an exon or in a 5′ or 3′ non-translated or non-transcribedregion. Such alterations include an alteration in a control element,e.g., a promoter, enhancer, and an alteration in a cis-acting ortrans-acting control element.

A template nucleic acid having homology with a target position in atarget gene may be used to alter the structure of a target sequence. Thetemplate sequence may be used to alter an unwanted structure, e.g., anunwanted or mutant nucleotide. The template nucleic acid may includesequence which, when integrated, results in: decreasing the activity ofa positive control element; increasing the activity of a positivecontrol element; decreasing the activity of a negative control element;increasing the activity of a negative control element; decreasing theexpression of a gene; increasing the expression of a gene; increasingresistance to a disorder or disease; increasing resistance to viralentry; correcting a mutation or altering an unwanted amino acid residueconferring, increasing, abolishing or decreasing a biological propertyof a gene product, e.g., increasing the enzymatic activity of an enzyme,or increasing the ability of a gene product to interact with anothermolecule.

The template nucleic acid may include sequence which results in: achange in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12 or morenucleotides of the target sequence.

A template polynucleotide may be of any suitable length, such as aboutor more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, ormore nucleotides in length. In an embodiment, the template nucleic acidmay be 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/-10, 70+/- 10, 80+/-10, 90+/- 10, 100+/- 10, 1 10+/- 10, 120+/- 10, 130+/- 10, 140+/- 10,150+/-10, 160+/- 10, 170+/- 10, 1 80+/- 10, 190+/- 10, 200+/- 10,210+/-10, of 220+/- 10 nucleotides in length. In an embodiment, thetemplate nucleic acid may be 30+/-20, 40+/-20, 50+/-20, 60+/-20, 70+/-20, 80+/-20, 90+/-20, 100+/-20, 1 10+/-20, 120+/-20, 130+/-20, 140+/-20,150+/-20, 160+/-20, 170+/-20, 180+/-20, 190+/-20, 200+/-20, 210+/-20, of220+/-20 nucleotides in length. In an embodiment, the template nucleicacid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to500, 50 to 400, 50 to300, 50 to 200, or 50 to 100 nucleotides in length.

In some embodiments, the template polynucleotide is complementary to aportion of a polynucleotide comprising the target sequence. Whenoptimally aligned, a template polynucleotide might overlap with one ormore nucleotides of a target sequences (e.g., about or more than about1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or morenucleotides). In some embodiments, when a template sequence and apolynucleotide comprising a target sequence are optimally aligned, thenearest nucleotide of the template polynucleotide is within about 1, 5,10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, ormore nucleotides from the target sequence.

The exogenous polynucleotide template comprises a sequence to beintegrated (e.g., a mutated gene). The sequence for integration may be asequence endogenous or exogenous to the cell. Examples of a sequence tobe integrated include polynucleotides encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000.

An upstream or downstream sequence may comprise from about 20 bp toabout 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplaryupstream or downstream sequence have about 200 bp to about 2000 bp,about 600 bp to about 1000 bp, or more particularly about 700 bp toabout 1000.

In certain embodiments, one or both homology arms may be shortened toavoid including certain sequence repeat elements. For example, a 5′homology arm may be shortened to avoid a sequence repeat element. Inother embodiments, a 3′ homology arm may be shortened to avoid asequence repeat element. In some embodiments, both the 5′ and the 3′homology arms may be shortened to avoid including certain sequencerepeat elements.

In some methods, the exogenous polynucleotide template may furthercomprise a marker. Such a marker may make it easy to screen for targetedintegrations. Examples of suitable markers include restriction sites,fluorescent proteins, or selectable markers. The exogenouspolynucleotide template of the disclosure can be constructed usingrecombinant techniques (see, for example, Sambrook et al., 2001 andAusubel et al., 1996).

In certain embodiments, a template nucleic acid for correcting amutation may be designed for use as a single-stranded oligonucleotide.When using a single-stranded oligonucleotide, 5′ and 3′ homology armsmay range up to about 200 base pairs (bp) in length, e.g., at least 25,50, 75, 100, 125, 150, 175, or 200 bp in length.

In certain embodiments, a template nucleic acid for correcting amutation may be designed for use with a homology-independent targetedintegration system. Suzuki et al. describe in vivo genome editing viaCRISPR/Cas9 mediated homology-independent targeted integration (2016,Nature 540:144-149). Schmid-Burgk, et al. describe use of theCRISPR-Cas9 system to introduce a double-strand break (DSB) at auser-defined genomic location and insertion of a universal donor DNA(Nat Commun. 2016 Jul 28;7:12338). Gao, et al. describe “Plug-and-PlayProtein Modification Using Homology-Independent Universal GenomeEngineering” (Neuron. 2019 Aug 21;103(4):583-597).

RNAi

In some embodiments, the genetic modifying agents may be interferingRNAs. In certain embodiments, diseases caused by a dominant mutation ina gene is targeted by silencing the mutated gene using RNAi. In somecases, the nucleotide sequence may comprise coding sequence for one ormore interfering RNAs. In certain examples, the nucleotide sequence maybe interfering RNA (RNAi). As used herein, the term “RNAi” refers to anytype of interfering RNA, including but not limited to, siRNAi, shRNAi,endogenous microRNA and artificial microRNA. For instance, it includessequences previously identified as siRNA, regardless of the mechanism ofdown-stream processing of the RNA (i.e., although siRNAs are believed tohave a specific method of in vivo processing resulting in the cleavageof mRNA, such sequences can be incorporated into the vectors in thecontext of the flanking sequences described herein). The term “RNAi” caninclude both gene silencing RNAi molecules, and also RNAi effectormolecules which activate the expression of a gene.

In certain embodiments, a modulating agent may comprise silencing one ormore endogenous genes. As used herein, “gene silencing” or “genesilenced” in reference to an activity of an RNAi molecule, for example asiRNA or miRNA refers to a decrease in the mRNA level in a cell for atarget gene by at least about 5%, about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%,about 99%, about 100% of the mRNA level found in the cell without thepresence of the miRNA or RNA interference molecule. In one preferredembodiment, the mRNA levels are decreased by at least about 70%, about80%, about 90%, about 95%, about 99%, about 100%.

As used herein, a “siRNA” refers to a nucleic acid that forms a doublestranded RNA, which double stranded RNA has the ability to reduce orinhibit expression of a gene or target gene when the siRNA is present orexpressed in the same cell as the target gene. The double stranded RNAsiRNA can be formed by the complementary strands. In one embodiment, asiRNA refers to a nucleic acid that can form a double stranded siRNA.The sequence of the siRNA can correspond to the full-length target gene,or a subsequence thereof. Typically, the siRNA is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded siRNA is about 15-50 nucleotides in length, and the doublestranded siRNA is about 15-50 base pairs in length, preferably about19-30 base nucleotides, preferably about 20-25 nucleotides in length,e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength).

As used herein “shRNA” or “small hairpin RNA” (also called stem loop) isa type of siRNA. In one embodiment, these shRNAs are composed of ashort, e.g., about 19 to about 25 nucleotide, antisense strand, followedby a nucleotide loop of about 5 to about 9 nucleotides, and theanalogous sense strand. Alternatively, the sense strand can precede thenucleotide loop structure and the antisense strand can follow.

The terms “microRNA” or “miRNA” are used interchangeably herein areendogenous RNAs, some of which are known to regulate the expression ofprotein-coding genes at the posttranscriptional level. EndogenousmicroRNAs are small RNAs naturally present in the genome that arecapable of modulating the productive utilization of mRNA. The termartificial microRNA includes any type of RNA sequence, other thanendogenous microRNA, which is capable of modulating the productiveutilization of mRNA. MicroRNA sequences have been described inpublications such as Lim, et al., Genes & Development, 17, p. 991 - 1008(2003), Lim et al Science 299, 1540 (2003), Lee and Ambros Science, 294,862 (2001), Lau et al., Science 294, 858-861 (2001), Lagos-Quintana etal, Current Biology, 12, 735-739 (2002), Lagos Quintana et al, Science294, 853- 857 (2001), and Lagos-Quintana et al, RNA, 9, 175- 179 (2003),which are incorporated by reference. Multiple microRNAs can also beincorporated into a precursor molecule. Furthermore, miRNA-likestem-loops can be expressed in cells as a vehicle to deliver artificialmiRNAs and short interfering RNAs (siRNAs) for the purpose of modulatingthe expression of endogenous genes through the miRNA and or RNAipathways.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat are comprised of two strands. Double-stranded molecules includethose comprised of a single RNA molecule that doubles back on itself toform a two-stranded structure. For example, the stem loop structure ofthe progenitor molecules from which the single-stranded miRNA isderived, called the pre-miRNA (Bartel et al. 2004. Cell 1 16:281 -297),comprises a dsRNA molecule.

Administration of Therapeutic Agents

For therapeutic uses, the compositions or agents described herein may beadministered systemically, for example, formulated in apharmaceutically-acceptable buffer such as physiological saline.Preferable routes of administration include, for example, subcutaneous,intravenous, interperitoneal, intramuscular, or intradermal injectionsthat provide continuous, sustained levels of the drug in the patient.Treatment of human patients or other animals will be carried out using atherapeutically effective amount of a therapeutic identified herein in aphysiologically-acceptable carrier. Suitable carriers and theirformulation are described, for example, in Remington’s PharmaceuticalSciences by E. W. Martin. The amount of the therapeutic agent to beadministered varies depending upon the manner of administration, the ageand body weight of the patient, and with the clinical symptoms of theneoplasia. Generally, amounts will be in the range of those used forother agents used in the treatment of other diseases associated withneoplasia, although in certain instances lower amounts will be neededbecause of the increased specificity of the compound. For example, atherapeutic compound is administered at a dosage that is cytotoxic to aneoplastic cell.

Human dosage amounts can initially be determined by extrapolating fromthe amount of compound used in mice, as a skilled artisan recognizes itis routine in the art to modify the dosage for humans compared to animalmodels. In certain embodiments, it is envisioned that the dosage mayvary from between about 1 µg compound/Kg body weight to about 5000 mgcompound/Kg body weight; or from about 5 mg/Kg body weight to about 4000mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kgbody weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg bodyweight; or from about 100 mg/Kg body weight to about 1000 mg/Kg bodyweight; or from about 150 mg/Kg body weight to about 500 mg/Kg bodyweight. In other cases, this dose may be about 1, 5, 10, 25, 50, 75,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000,4500, or 5000 mg/Kg body weight. In other aspects, it is envisaged thatdoses may be in the range of about 5 mg compound/Kg body to about 20 mgcompound/Kg body. In other embodiments, the doses may be about 8, 10,12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may beadjusted upward or downward, as is routinely done in such treatmentprotocols, depending on the results of the initial clinical trials andthe needs of a particular patient.

In some cases, the compound or composition of the invention isadministered at a dose that is lower than the human equivalent dosage(HED) of the no observed adverse effect level (NOAEL) over a period ofthree months, four months, six months, nine months, 1 year, 2 years, 3years, 4 years or more. The NOAEL, as determined in animal studies, isuseful in determining the maximum recommended starting dose for humanclinical trials. For instance, the NOAELs can be extrapolated todetermine human equivalent dosages. Typically, such extrapolationsbetween species are conducted based on the doses that are normalized tobody surface area (i.e., mg/m²). In specific embodiments, the NOAELs aredetermined in mice, hamsters, rats, ferrets, guinea pigs, rabbits, dogs,primates, primates (monkeys, marmosets, squirrel monkeys, baboons),micropigs or minipigs. For a discussion on the use of NOAELs and theirextrapolation to determine human equivalent doses, see Guidance forIndustry Estimating the Maximum Safe Starting Dose in Initial ClinicalTrials for Therapeutics in Adult Healthy Volunteers, U.S. Department ofHealth and Human Services Food and Drug Administration Center for DrugEvaluation and Research (CDER), Pharmacology and Toxicology, July 2005,incorporated herein by reference.

The amount of an agent of the invention used in the prophylactic and/ortherapeutic regimens which will be effective in the prevention,treatment, and/or management of cancer can be based on the currentlyprescribed dosage of the agent as well as assessed by methods disclosedherein and known in the art. The frequency and dosage will vary alsoaccording to factors specific for each patient depending on the specificcompounds administered, the severity of the cancerous condition, theroute of administration, as well as age, body, weight, response, and thepast medical history of the patient. For example, the dosage of an agentof the invention which will be effective in the treatment, prevention,and/or management of cancer can be determined by administering thecompound to an animal model such as, e.g., the animal models disclosedherein or known to those skilled in the art. In addition, in vitroassays may optionally be employed to help identify optimal dosageranges.

In some aspects, the prophylactic and/or therapeutic regimens comprisetitrating the dosages administered to the patient so as to achieve aspecified measure of therapeutic efficacy. Such measures include areduction in the cancer cell population in the patient.

In certain cases, the dosage of the compound of the invention in theprophylactic and/or therapeutic regimen is adjusted so as to achieve areduction in the number or amount of cancer cells found in a testspecimen extracted from a patient after undergoing the prophylacticand/or therapeutic regimen, as compared with a reference sample. Here,the reference sample is a specimen extracted from the patient undergoingtherapy, wherein the specimen is extracted from the patient at anearlier time point. In one aspect, the reference sample is a specimenextracted from the same patient, prior to receiving the prophylacticand/or therapeutic regimen. For example, the number or amount of cancercells in the test specimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 99% lower than in the reference sample.

In some cases, the dosage of the compound of the invention in theprophylactic and/or therapeutic regimen is adjusted so as to achieve anumber or amount of cancer cells that falls within a predeterminedreference range. In these embodiments, the number or amount of cancercells in a test specimen is compared with a predetermined referencerange.

In other embodiments, the dosage of the compound of the invention inprophylactic and/or therapeutic regimen is adjusted so as to achieve areduction in the number or amount of cancer cells found in a testspecimen extracted from a patient after undergoing the prophylacticand/or therapeutic regimen, as compared with a reference sample, whereinthe reference sample is a specimen is extracted from a healthy,noncancer-afflicted patient. For example, the number or amount of cancercells in the test specimen is at least within 60%, 50%, 40%, 30%, 20%,15%, 10%, 5%, or 2% of the number or amount of cancer cells in thereference sample.

In treating certain human patients having solid tumors, extractingmultiple tissue specimens from a suspected tumor site may proveimpracticable. In these cases, the dosage of the compounds of theinvention in the prophylactic and/or therapeutic regimen for a humanpatient is extrapolated from doses in animal models that are effectiveto reduce the cancer population in those animal models. In the animalmodels, the prophylactic and/or therapeutic regimens are adjusted so asto achieve a reduction in the number or amount of cancer cells found ina test specimen extracted from an animal after undergoing theprophylactic and/or therapeutic regimen, as compared with a referencesample. The reference sample can be a specimen extracted from the sameanimal, prior to receiving the prophylactic and/or therapeutic regimen.In specific embodiments, the number or amount of cancer cells in thetest specimen is at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50% or 60%lower than in the reference sample. The doses effective in reducing thenumber or amount of cancer cells in the animals can be normalized tobody surface area (e.g., mg/m²) to provide an equivalent human dose.

The prophylactic and/or therapeutic regimens disclosed herein compriseadministration of compounds of the invention or pharmaceuticalcompositions thereof to the patient in a single dose or in multipledoses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, or more doses).

In one aspect, the prophylactic and/or therapeutic regimens compriseadministration of the compounds of the invention or pharmaceuticalcompositions thereof in multiple doses. When administered in multipledoses, the compounds or pharmaceutical compositions are administeredwith a frequency and in an amount sufficient to prevent, treat, and/ormanage the condition. For example, the frequency of administrationranges from once a day up to about once every eight weeks. In anotherexample, the frequency of administration ranges from about once a weekup to about once every six weeks. In another example, the frequency ofadministration ranges from about once every three weeks up to about onceevery four weeks.

Generally, the dosage of a compound of the invention administered to asubject to prevent, treat, and/or manage cancer is in the range of 0.01to 500 mg/kg, e.g., in the range of 0.1 mg/kg to 100 mg/kg, of thesubject’s body weight. For example, the dosage administered to a subjectis in the range of 0.1 mg/kg to 50 mg/kg, or 1 mg/kg to 50 mg/kg, of thesubject’s body weight, more preferably in the range of 0.1 mg/kg to 25mg/kg, or 1 mg/kg to 25 mg/kg, of the patient’s body weight. In anotherexample, the dosage of a compound of the invention administered to asubject to prevent, treat, and/or manage cancer in a patient is 500mg/kg or less, preferably 250 mg/kg or less, 100 mg/kg or less, 95 mg/kgor less, 90 mg/kg or less, 85 mg/kg or less, 80 mg/kg or less, 75 mg/kgor less, 70 mg/kg or less, 65 mg/kg or less, 60 mg/kg or less, 55 mg/kgor less, 50 mg/kg or less, 45 mg/kg or less, 40 mg/kg or less, 35 mg/kgor less, 30 mg/kg or less, 25 mg/kg or less, 20 mg/kg or less, 15 mg/kgor less, 10 mg/kg or less, 5 mg/kg or less, 2.5 mg/kg or less, 2 mg/kgor less, 1.5 mg/kg or less, or 1 mg/kg or less of a patient’s bodyweight.

In another example, the dosage of a compound of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is a unit dose of 0.1 to 50 mg, 0.1 mg to 20 mg, 0.1 mg to 15mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg,0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to12 mg, 0.25 to 10 mg, 0.25 to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg,0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

In another example, the dosage of a compound of the inventionadministered to a subject to prevent, treat, and/or manage cancer in apatient is in the range of 0.01 to 10 g/m², and more typically, in therange of 0.1 g/m² to 7.5 g/m², of the subject’s body weight. Forexample, the dosage administered to a subject is in the range of 0.5g/m² to 5 g/m², or 1 g/m² to 5 g/m² of the subject’s body’s surfacearea.

In another example, the prophylactic and/or therapeutic regimencomprises administering to a patient one or more doses of an effectiveamount of a compound of the invention, wherein the dose of an effectiveamount achieves a plasma level of at least 0.1 µg/mL, at least 0.5µg/mL, at least 1 µg/mL, at least 2 µg/mL, at least 5 µg/mL, at least 6µg/mL, at least 10 µg/mL, at least 15 µg/mL, at least 20 µg/mL, at least25 µg/mL, at least 50 µg/mL, at least 100 µg/mL, at least 125 µg/mL, atleast 150 µg/mL, at least 175 µg/mL, at least 200 µg/mL, at least 225µg/mL, at least 250 µg/mL, at least 275 µg/mL, at least 300 µg/mL, atleast 325 µg/mL, at least 350 µg/mL, at least 375 µg/mL, or at least 400µg/mL of the compound of the invention.

In another example, the prophylactic and/or therapeutic regimencomprises administering to a patient a plurality of doses of aneffective amount of a compound of the invention, wherein the pluralityof doses maintains a plasma level of at least 0.1 µg/mL, at least 0.5µg/mL, at least 1 µg/mL, at least 2 µg/mL, at least 5 µg/mL, at least 6µg/mL, at least 10 µg/mL, at least 15 µg/mL, at least 20 µg/mL, at least25 µg/mL, at least 50 µg/mL, at least 100 µg/mL, at least 125 µg/mL, atleast 150 µg/mL, at least 175 µg/mL, at least 200 µg/mL, at least 225µg/mL, at least 250 µg/mL, at least 275 µg/mL, at least 300 µg/mL, atleast 325 µg/mL, at least 350 µg/mL, at least 375 µg/mL, or at least 400µg/mL of the compound of the invention for at least 1 day, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 monthsor 36 months.

In other embodiments, the prophylactic and/or therapeutic regimencomprises administering to a patient a plurality of doses of aneffective amount of a compound of the invention, wherein the pluralityof doses maintains a plasma level of at least 0.1 µg/mL, at least 0.5µg/mL, at least 1 µg/mL, at least 2 µg/mL, at least 5 µg/mL, at least 6µg/mL, at least 10 µg/mL, at least 15 µg/mL, at least 20 µg/mL, at least25 µg/mL, at least 50 µg/mL, at least 100 µg/mL, at least 125 µg/mL, atleast 150 µg/mL, at least 175 µg/mL, at least 200 µg/mL, at least 225µg/mL, at least 250 µg/mL, at least 275 µg/mL, at least 300 µg/mL, atleast 325 µg/mL, at least 350 µg/mL, at least 375 µg/mL, or at least 400µg/mL of the compound of the invention for at least 1 day, 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 15 months, 18 months, 24 monthsor 36 months.

Release of Pharmaceutical Compositions

Pharmaceutical compositions according to the invention may be formulatedto release the active compound substantially immediately uponadministration or at any predetermined time or time period afteradministration. The latter types of compositions are generally known ascontrolled release formulations, which include (i) formulations thatcreate a substantially constant concentration of the drug within thebody over an extended period of time; (ii) formulations that after apredetermined lag time create a substantially constant concentration ofthe drug within the body over an extended period of time; (iii)formulations that sustain action during a predetermined time period bymaintaining a relatively, constant, effective level in the body withconcomitant minimization of undesirable side effects associated withfluctuations in the plasma level of the active substance (sawtoothkinetic pattern); (iv) formulations that localize action by, e.g.,spatial placement of a controlled release composition adjacent to or incontact with the thymus; (v) formulations that allow for convenientdosing, such that doses are administered, for example, once every one ortwo weeks; and (vi) formulations that target a neoplasia by usingcarriers or chemical derivatives to deliver the therapeutic agent to aparticular cell type (e.g., neoplastic cell). For some applications,controlled release formulations obviate the need for frequent dosingduring the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtaincontrolled release in which the rate of release outweighs the rate ofmetabolism of the compound in question. In one example, controlledrelease is obtained by appropriate selection of various formulationparameters and ingredients, including, e.g., various types of controlledrelease compositions and coatings. Thus, the therapeutic is formulatedwith appropriate excipients into a pharmaceutical composition that, uponadministration, releases the therapeutic in a controlled manner.Examples include single or multiple unit tablet or capsule compositions,oil solutions, suspensions, emulsions, microcapsules, microspheres,molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent that reduces orameliorates a neoplasia, the composition may include suitableparenterally acceptable carriers and/or excipients. The activetherapeutic agent(s) may be incorporated into microspheres,microcapsules, nanoparticles, liposomes, or the like for controlledrelease. Furthermore, the composition may include suspending,solubilizing, stabilizing, pH-adjusting agents, tonicity adjustingagents, and/or dispersing, agents.

As indicated above, the pharmaceutical compositions according to theinvention may be in the form suitable for sterile injection. To preparesuch a composition, the suitable active antineoplastic therapeutic(s)are dissolved or suspended in a parenterally acceptable liquid vehicle.Among acceptable vehicles and solvents that may be employed are water,water adjusted to a suitable pH by addition of an appropriate amount ofhydrochloric acid, sodium hydroxide or a suitable buffer,1,3-butanediol, Ringer’s solution, and isotonic sodium chloride solutionand dextrose solution. The aqueous formulation may also contain one ormore preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).In cases where one of the compounds is only sparingly or slightlysoluble in water, a dissolution enhancing or solubilizing agent can beadded, or the solvent may include 10-60% w/w of propylene glycol.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, or emulsions. Alternatively, the active drugmay be incorporated in biocompatible carriers, liposomes, nanoparticles,implants, or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam- nine) and, poly(lactic acid).Biocompatible carriers that may be used when formulating a controlledrelease parenteral formulation are carbohydrates (e.g., dextrans),proteins (e.g., albumin), lipoproteins, or antibodies. Materials for usein implants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Vector Delivery

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.Delivery vehicles, vectors, particles, nanoparticles, formulations andcomponents thereof for expression of one or more elements of a nucleicacid-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667).

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Ribonucleoprotein (RNP)

In particular embodiments, pre-complexed guide RNA and CRISPR effectorprotein, (optionally, adenosine deaminase fused to a CRISPR protein oran adaptor) are delivered as a ribonucleoprotein (RNP). RNPs have theadvantage that they lead to rapid editing effects even more so than theRNA method because this process avoids the need for transcription. Animportant advantage is that both RNP delivery is transient, reducingoff-target effects and toxicity issues. Efficient genome editing indifferent cell types has been observed by Kim et al. (2014, Genome Res.24(6):1012-9), Paix et al. (2015, Genetics 204(1):47-54), Chu et al.(2016, BMC Biotechnol. 16:4), and Wang et al. (2013, Cell.9;153(4):910-8).

In particular embodiments, the ribonucleoprotein is delivered by way ofa polypeptide-based shuttle agent as described in WO2016161516.WO2016161516 describes efficient transduction of polypeptide cargosusing synthetic peptides comprising an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), to a histidine-richdomain and a CPD. Similarly, these polypeptides can be used for thedelivery of CRISPR-effector based RNPs in eukaryotic cells.

Administration of Proteins

Significant progress has been made in understanding pharmacokinetics(PK), pharmacodynamics (PD), as well as toxicity profiles of therapeuticproteins in animals and humans, which have been in commercialdevelopment for more than three decades (see, e.g., Vugmeyster et al.,Pharmacokinetics and toxicology of therapeutic proteins: Advances andchallenges, World J Biol Chem. 2012 Apr 26; 3(4): 73-92). In certainembodiments, therapeutic proteins are administered by parenteral routes,such as intravenous (IV), subcutaneous (SC) or intramuscular (IM)injection. Molecular size, hydrophilicity, and gastric degradation arethe main factors that preclude gastrointestinal (GI) absorption oftherapeutic proteins (see, e.g., Keizer, et al., Clinicalpharmacokinetics of therapeutic monoclonal antibodies. ClinPharmacokinet. 2010 Aug; 49(8):493-507). Pulmonary delivery with aerosolformulations or dry powder inhalers has been used for selected proteins,e.g., exubera (TM) (see, e.g., Scheuch and Siekmeier, Novel approachesto enhance pulmonary delivery of proteins and peptides. J PhysiolPharmacol. 2007 Nov; 58 Suppl 5(Pt 2):615-25). Intravitreal injectionshave been used for peptides and proteins that require only localactivity (see, e.g., Suresh, et al., Ocular Delivery of Peptides andProteins. In: Van Der Walle C., editor. Peptide and Protein Delivery.London: Academic Press; 2011. pp. 87-103). In certain embodiments, SCadministration of therapeutic proteins is often a preferred route. Inparticular, the suitability of SC dosing for self-administrationtranslates into significantly reduced treatment costs.

Standard of Care

Aspects of the invention involve modifying the therapy within a standardof care based on the detection of any of the biomarkers as describedherein. In one embodiment, therapy comprising an agent is administeredwithin a standard of care where addition of the agent is synergisticwithin the steps of the standard of care. In one embodiment, the agenttargets and/or shifts a tumor to be more vulnerable to a therapeuticagent targeting XPR1:KIDINS220-mediated phosphate export (e.g., aninhibitor). In one embodiment, the agent inhibits expression or activityof one or more genes involved in phosphate homeostasis. The term“standard of care” as used herein refers to the current treatment thatis accepted by medical experts as a proper treatment for a certain typeof disease and that is widely used by healthcare professionals. Standardof care is also called best practice, standard medical care, andstandard therapy. Standards of care for cancer generally includesurgery, lymph node removal, radiation, chemotherapy, targetedtherapies, antibodies targeting the tumor, and immunotherapy.Immunotherapy can include checkpoint blockers (CBP), chimeric antigenreceptors (CARs), and adoptive T-cell therapy. The standards of care forthe most common cancers can be found on the website of National CancerInstitute (www.cancer.gov/cancertopics). A treatment clinical trial is aresearch study meant to help improve current treatments or obtaininformation on new treatments for patients with cancer. When clinicaltrials show that a new treatment is better than the standard treatment,the new treatment may be considered the new standard treatment.

In certain embodiments, the present invention provides for one or moretherapeutic agents (e.g., inhibitors) that can be used in combinationwith the standard of care for the cancer. TargetingXPR1:KIDINS220-mediated phosphate export in combination within astandard of care may provide for enhanced or otherwise previouslyunknown activity in the treatment of disease.

In certain embodiments, the present invention provides for a combinationtherapy comprising a treatment described herein with a treatment that ispart of the standard of care for a cancer (i.e., a therapeutic regime).In certain embodiments, the standard of care for treating ovarian cancercomprises surgery, chemotherapy, and targeted therapy (see, e.g.,Lheureux et al., Epithelial ovarian cancer: Evolution of management inthe era of precision medicine. CA Cancer J Clin. 2019Jul;69(4):280-304). Ovarian cancer is a cancer that forms in or on anovary. Symptoms may include bloating, pelvic pain, abdominal swelling,and loss of appetite, among others. Common areas to which the cancer mayspread include the lining of the abdomen, lymph nodes, lungs, and liver.The most common type of ovarian cancer is ovarian carcinoma (>95% of allcases). There are five main subtypes of ovarian carcinoma, of whichhigh-grade serous carcinoma is the most common. These tumors arebelieved to start in the cells covering the ovaries, though some mayform at the Fallopian tubes. Less common types of ovarian cancer includegerm cell tumors and sex cord stromal tumors. A diagnosis of ovariancancer is confirmed through a biopsy of tissue, usually removed duringsurgery.

If caught and treated in an early stage, ovarian cancer is oftencurable. Treatment usually includes some combination of surgery,radiation therapy, and chemotherapy. Outcomes depend on the extent ofthe disease, the subtype of cancer present, and other medicalconditions. The overall five-year survival rate in the United States is45%.

If ovarian cancer recurs, it is considered partially platinum-sensitiveor platinum-resistant, based on the time since the last recurrencetreated with platins: partially platinum-sensitive cancers recurred 6-12months after last treatment, and platinum-resistant cancers have aninterval of less than 6 months.

For platinum-sensitive tumors, platins are utilized for second-linechemotherapy, often in combination with other cytotoxic agents. Regimensinclude carboplatin combined with pegylated liposomal doxorubicin,gemcitabine, or paclitaxel. If the tumor is determined to beplatinum-resistant, vincristine, dactinomycin, and cyclophosphamide(VAC) or some combination of paclitaxel, gemcitabine, and oxaliplatincan be used as a second-line therapy.

Systemic therapy can include single to combination chemotherapyapproaches alone or in combination with targeted therapy. In certainembodiments, surgery includes surgery for accurate surgical staging,primary debulking surgery, interval debulking surgery, and secondarydebulking surgery. In certain embodiments, chemotherapy includescarboplatin, cisplatin and paclitaxel. In certain embodiments, targetedtherapy includes Bevacizumab, which is a humanized monoclonal antibodyagainst vascular endothelial growth factor (VEGF), and poly (ADP-ribose)polymerase (PARP) inhibitors (e.g., Olaparib, Niraparib, and Rucaparib).Other therapies that may be used in combination with the presentinvention include agents targeting the folate receptor (e.g.,mirvetuximab soravtansine (IMGN853), which is an ADC consisting of ananti-FRα antibody linked to the tubulin-disrupting maytansinoid DM4drug, a potent antimitotic agent). In certain embodiments, checkpointblockade therapy is used in a combination therapy. As used herein,checkpoint blockade therapy (CPB) refers to antibodies that block theactivity of checkpoint receptors, including CTLA-4, PD-1, Tim-3, Lag-3,and TIGIT, either alone or in combination. The checkpoint blockadetherapy may comprise anti-TIM3, anti-CTLA4, anti-PD-L1, anti-PD1,anti-TIGIT, anti-LAG3, or combinations thereof. Anti-PD1 antibodies aredisclosed in U.S. Pat. No. 8,735,553. Antibodies to LAG-3 are disclosedin U.S. Pat. No. 9,132,281. Anti-CTLA4 antibodies are disclosed in U.S.Pat. No. 9,327,014; U.S. Pat. No. 9,320,811; and U.S. Pat. No.9,062,111. Specific check point inhibitors include, but are not limitedto, anti-CTLA4 antibodies (e.g., Ipilimumab and Tremelimumab), anti-PD-1antibodies (e.g., Nivolumab, Pembrolizumab), and anti-PD-L1 antibodies(e.g., Atezolizumab). In certain embodiments, chemotherapy incombination with immunotherapy is used in the treatment of ovariancancer. In certain embodiments, the combination therapy comprisespaclitaxel plus pembrolizumab, preferably in patients withplatinum-resistant ovarian cancer. In certain embodiments, thecombination therapy comprises immunotherapy combined with PARPinhibitors.

In one example, therapeutic agents are administered in a combinationtherapy, i.e., combined with other agents, e.g., therapeutic agents,that are useful for treating pathological conditions or disorders, suchas various forms of cancer. The term “in combination” in this contextmeans that the agents are given substantially contemporaneously, eithersimultaneously or sequentially. If given sequentially, at the onset ofadministration of the second agent, the first of the two agents is insome cases still detectable at effective concentrations at the site oftreatment.

The administration of an agent or a combination of agents for thetreatment of a neoplasia may be by any suitable means that results in aconcentration of the therapeutic that, combined with other components,is effective in ameliorating, reducing, or stabilizing a neoplasia. Theagent may be contained in any appropriate amount in any suitable carriersubstance, and is generally present in an amount of 1-95% by weight ofthe total weight of the composition. The composition may be provided ina dosage form that is suitable for parenteral (e.g., subcutaneously,intravenously, intramuscularly, or intraperitoneally) administrationroute. The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20^(th) ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Accordingly, in some examples, the prophylactic and/or therapeuticregimen comprises administration of an agent of the invention incombination with one or more additional anticancer therapeutics. In oneexample, the dosages of the one or more additional anticancertherapeutics used in the combination therapy is lower than those whichhave been or are currently being used to prevent, treat, and/or managecancer. The recommended dosages of the one or more additional anticancertherapeutics currently used for the prevention, treatment, and/ormanagement of cancer can be obtained from any reference in the artincluding, but not limited to, Hardman et al., eds., Goodman & Gilman’sThe Pharmacological Basis of Therapeutics, 10^(th) ed., McGraw-Hill, NewYork, 2001; Physician’s Desk Reference (60^(th) ed., 2006), which isincorporated herein by reference in its entirety.

The agent of the invention and the one or more additional anticancertherapeutics can be administered separately, simultaneously, orsequentially. In various aspects, the agent of the invention and theadditional anticancer therapeutic are administered less than 5 minutesapart, less than 30 minutes apart, less than 1 hour apart, at about 1hour apart, at about 1 to about 2 hours apart, at about 2 hours to about3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hoursto about 5 hours apart, at about 5 hours to about 6 hours apart, atabout 6 hours to about 7 hours apart, at about 7 hours to about 8 hoursapart, at about 8 hours to about 9 hours apart, at about 9 hours toabout 10 hours apart, at about 10 hours to about 11 hours apart, atabout 11 hours to about 12 hours apart, at about 12 hours to 18 hoursapart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hoursto 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hoursapart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hoursto 96 hours apart, or 96 hours to 120 hours part. In another example,two or more anticancer therapeutics are administered within the samepatient visit.

In certain aspects, the agent of the invention and the additionalanticancer therapeutic are cyclically administered. Cycling therapyinvolves the administration of one anticancer therapeutic for a periodof time, followed by the administration of a second anticancertherapeutic for a period of time and repeating this sequentialadministration, i.e., the cycle, in order to reduce the development ofresistance to one or both of the anticancer therapeutics, to avoid orreduce the side effects of one or both of the anticancer therapeutics,and/or to improve the efficacy of the therapies. In one example, cyclingtherapy involves the administration of a first anticancer therapeuticfor a period of time, followed by the administration of a secondanticancer therapeutic for a period of time, optionally, followed by theadministration of a third anticancer therapeutic for a period of timeand so forth, and repeating this sequential administration, i.e., thecycle in order to reduce the development of resistance to one of theanticancer therapeutics, to avoid or reduce the side effects of one ofthe anticancer therapeutics, and/or to improve the efficacy of theanticancer therapeutics.

In another example, the anticancer therapeutics are administeredconcurrently to a subj ect in separate compositions. The combinationanticancer therapeutics of the invention may be administered to asubject by the same or different routes of administration.

When an agent of the invention and the additional anticancer therapeuticare administered to a subject concurrently, the term “concurrently” isnot limited to the administration of the anticancer therapeutics atexactly the same time, but rather, it is meant that they areadministered to a subject in a sequence and within a time interval suchthat they can act together (e.g., synergistically to provide anincreased benefit than if they were administered otherwise). Forexample, the anticancer therapeutics may be administered at the sametime or sequentially in any order at different points in time; however,if not administered at the same time, they should be administeredsufficiently close in time so as to provide the desired therapeuticeffect, preferably in a synergistic fashion. The combination anticancertherapeutics of the invention can be administered separately, in anyappropriate form and by any suitable route. When the components of thecombination anticancer therapeutics are not administered in the samepharmaceutical composition, it is understood that they can beadministered in any order to a subject in need thereof. For example, aagent of the invention can be administered prior to (e.g., 5 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks,4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantlywith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks after) the administration of the additionalanticancer therapeutic, to a subject in need thereof. In variousaspects, the anticancer therapeutics are administered 1 minute apart, 10minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hoursapart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hoursto 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hoursapart, no more than 24 hours apart or no more than 48 hours apart. Inone example, the anticancer therapeutics are administered within thesame office visit. In another example, the combination anticancertherapeutics of the invention are administered at 1 minute to 24 hoursapart.

Diagnostic Methods

The invention provides biomarkers (e.g., malignant cell specificmarkers) for the identification, diagnosis, prognosis and manipulationof cell properties, for use in a variety of diagnostic and/ortherapeutic indications. In certain embodiments, the marker may beSLC34A2, SLC20A1, FGF23 and/or PAX8. In certain embodiments, the markeris a gene that covaries with SLC34A2 (e.g., PAX8) and thus can bedetected in combination or alone. In certain embodiments, detecting atumor marker may indicate that a subject suffering from a cancer mayrespond to inhibition of XPR1:KIDINS220-mediated phosphate export. Incertain embodiments, detecting a tumor marker may indicate prognosis fora subject suffering from cancer. In certain embodiments, detecting atumor marker may indicate a treatment is effective (i.e., monitoring theefficacy of the treatment). In certain embodiments, tumor cells thatexpress SLC34A2 higher than in normal tissue are vulnerable toinhibition of XPR1:KIDINS220-mediated phosphate export (see, therapeuticmethods). In certain embodiments, inhibition of XPR1:KIDINS220-mediatedphosphate export results in a decrease in SLC34A2 and/or SLC20A1expression, and/or an increase in FGF23 expression.

In certain embodiments, morphological changes can be used to determinewhether a treatment is effective. In certain embodiments, treatment of asubject with an inhibitor of XPR1:KIDINS220-mediated phosphate exportresults in an increase in vacuole-like structures in tumor cells. Incertain embodiments, the morphological changes are detected bymicroscopy.

In certain embodiments, an RBD protein as described in the therapeuticmethods can be used for detection of XPR1 expressing cells. Giovannini,et al., 2013 show monitoring cell-surface expression of XPR1 with asoluble ligand derived from X-MLV Env RBD (XRBD) See also, US 9,791,435B2; and US 2015/0099653 A1.

Biomarkers in the context of the present invention encompasses, withoutlimitation nucleic acids, proteins, reaction products, and metabolites,together with their polymorphisms, mutations, variants, modifications,subunits, fragments, and other analytes or sample-derived measures. Incertain embodiments, biomarkers include the signature genes or signaturegene products, and/or cells as described herein.

Biomarkers are useful in methods of diagnosing, prognosing and/orstaging an immune response in a subject by detecting a first level ofexpression, activity and/or function of one or more biomarker andcomparing the detected level to a control of level wherein a differencein the detected level and the control level indicates that the presenceof an immune response in the subject.

The terms “diagnosis” and “monitoring” are commonplace andwell-understood in medical practice. By means of further explanation andwithout limitation the term “diagnosis” generally refers to the processor act of recognising, deciding on or concluding on a disease orcondition in a subject on the basis of symptoms and signs and/or fromresults of various diagnostic procedures (such as, for example, fromknowing the presence, absence and/or quantity of one or more biomarkerscharacteristic of the diagnosed disease or condition).

The terms “prognosing” or “prognosis” generally refer to an anticipationon the progression of a disease or condition and the prospect (e.g., theprobability, duration, and/or extent) of recovery. A good prognosis ofthe diseases or conditions taught herein may generally encompassanticipation of a satisfactory partial or complete recovery from thediseases or conditions, preferably within an acceptable time period. Agood prognosis of such may more commonly encompass anticipation of notfurther worsening or aggravating of such, preferably within a given timeperiod. A poor prognosis of the diseases or conditions as taught hereinmay generally encompass anticipation of a substandard recovery and/orunsatisfactorily slow recovery, or to substantially no recovery or evenfurther worsening of such.

The biomarkers of the present invention are useful in methods ofidentifying patient populations at risk or suffering from cancer or foridentifying patients that will respond to specific treatments based on adetected level of expression, activity and/or function of one or morebiomarkers. These biomarkers are also useful in monitoring subjectsundergoing treatments and therapies for suitable or aberrant response(s)to determine efficaciousness of the treatment or therapy and forselecting or modifying therapies and treatments that would beefficacious in treating, delaying the progression of or otherwiseameliorating a symptom. The biomarkers provided herein are useful forselecting a group of patients at a specific state of a disease withaccuracy that facilitates selection of treatments.

The term “monitoring” generally refers to the follow-up of a disease ora condition in a subject for any changes which may occur over time.

The terms also encompass prediction of a disease. The terms “predicting”or “prediction” generally refer to an advance declaration, indication orforetelling of a disease or condition in a subject not (yet) having saiddisease or condition. For example, a prediction of a disease orcondition in a subject may indicate a probability, chance or risk thatthe subject will develop said disease or condition, for example within acertain time period or by a certain age. Said probability, chance orrisk may be indicated inter alia as an absolute value, range orstatistics, or may be indicated relative to a suitable control subjector subject population (such as, e.g., relative to a general, normal orhealthy subject or subject population). Hence, the probability, chanceor risk that a subject will develop a disease or condition may beadvantageously indicated as increased or decreased, or as fold-increasedor fold-decreased relative to a suitable control subject or subjectpopulation. As used herein, the term “prediction” of the conditions ordiseases as taught herein in a subject may also particularly mean thatthe subject has a ‘positive’ prediction of such, i.e., that the subjectis at risk of having such (e.g., the risk is significantly increasedvis-à-vis a control subject or subject population). The term “predictionof no” diseases or conditions as taught herein as described herein in asubject may particularly mean that the subject has a ‘negative’prediction of such, i.e., that the subject’s risk of having such is notsignificantly increased vis-à-vis a control subject or subjectpopulation.

Suitably, an altered quantity or phenotype of the immune cells in thesubject compared to a control subject having normal immune status or nothaving a disease comprising an immune component indicates that thesubject has an impaired immune status or has a disease comprising animmune component or would benefit from an immune therapy.

Hence, the methods may rely on comparing the quantity of immune cellpopulations, biomarkers, or gene or gene product signatures measured insamples from patients with reference values, wherein said referencevalues represent known predictions, diagnoses and/or prognoses ofdiseases or conditions as taught herein.

For example, distinct reference values may represent the prediction of arisk (e.g., an abnormally elevated risk) of having a given disease orcondition as taught herein vs. the prediction of no or normal risk ofhaving said disease or condition. In another example, distinct referencevalues may represent predictions of differing degrees of risk of havingsuch disease or condition.

In a further example, distinct reference values can represent thediagnosis of a given disease or condition as taught herein vs. thediagnosis of no such disease or condition (such as, e.g., the diagnosisof healthy, or recovered from said disease or condition, etc.). Inanother example, distinct reference values may represent the diagnosisof such disease or condition of varying severity.

In yet another example, distinct reference values may represent a goodprognosis for a given disease or condition as taught herein vs. a poorprognosis for said disease or condition. In a further example, distinctreference values may represent varyingly favourable or unfavourableprognoses for such disease or condition.

Such comparison may generally include any means to determine thepresence or absence of at least one difference and optionally of thesize of such difference between values being compared. A comparison mayinclude a visual inspection, an arithmetical or statistical comparisonof measurements. Such statistical comparisons include, but are notlimited to, applying a rule.

Reference values may be established according to known procedurespreviously employed for other cell populations, biomarkers and gene orgene product signatures. For example, a reference value may beestablished in an individual or a population of individualscharacterised by a particular diagnosis, prediction and/or prognosis ofsaid disease or condition (i.e., for whom said diagnosis, predictionand/or prognosis of the disease or condition holds true). Suchpopulation may comprise without limitation 2 or more, 10 or more, 100 ormore, or even several hundred or more individuals.

A “deviation” of a first value from a second value may generallyencompass any direction (e.g., increase: first value > second value; ordecrease: first value < second value) and any extent of alteration.

For example, a deviation may encompass a decrease in a first value by,without limitation, at least about 10% (about 0.9-fold or less), or byat least about 20% (about 0.8-fold or less), or by at least about 30%(about 0.7-fold or less), or by at least about 40% (about 0.6-fold orless), or by at least about 50% (about 0.5-fold or less), or by at leastabout 60% (about 0.4-fold or less), or by at least about 70% (about0.3-fold or less), or by at least about 80% (about 0.2-fold or less), orby at least about 90% (about 0.1-fold or less), relative to a secondvalue with which a comparison is being made.

For example, a deviation may encompass an increase of a first value by,without limitation, at least about 10% (about 1.1-fold or more), or byat least about 20% (about 1.2-fold or more), or by at least about 30%(about 1.3-fold or more), or by at least about 40% (about 1.4-fold ormore), or by at least about 50% (about 1.5-fold or more), or by at leastabout 60% (about 1.6-fold or more), or by at least about 70% (about1.7-fold or more), or by at least about 80% (about 1.8-fold or more), orby at least about 90% (about 1.9-fold or more), or by at least about100% (about 2-fold or more), or by at least about 150% (about 2.5-foldor more), or by at least about 200% (about 3-fold or more), or by atleast about 500% (about 6-fold or more), or by at least about 700%(about 8-fold or more), or like, relative to a second value with which acomparison is being made.

Preferably, a deviation may refer to a statistically significantobserved alteration. For example, a deviation may refer to an observedalteration which falls outside of error margins of reference values in agiven population (as expressed, for example, by standard deviation orstandard error, or by a predetermined multiple thereof, e.g., ±1×SD or±2×SD or ±3×SD, or ±1×SE or ±2×SE or ±3×SE). Deviation may also refer toa value falling outside of a reference range defined by values in agiven population (for example, outside of a range which comprises ≥40%,≥ 50%, ≥60%, ≥70%, ≥75% or ≥80% or ≥85% or ≥90% or ≥95% or even ≥100% ofvalues in said population).

In a further embodiment, a deviation may be concluded if an observedalteration is beyond a given threshold or cut-off. Such threshold orcut-off may be selected as generally known in the art to provide for achosen sensitivity and/or specificity of the prediction methods, e.g.,sensitivity and/or specificity of at least 50%, or at least 60%, or atleast 70%, or at least 80%, or at least 85%, or at least 90%, or atleast 95%.

For example, receiver-operating characteristic (ROC) curve analysis canbe used to select an optimal cut-off value of the quantity of a givenimmune cell population, biomarker or gene or gene product signatures,for clinical use of the present diagnostic tests, based on acceptablesensitivity and specificity, or related performance measures which arewell-known per se, such as positive predictive value (PPV), negativepredictive value (NPV), positive likelihood ratio (LR+), negativelikelihood ratio (LR-), Youden index, or similar.

In one embodiment, the signature genes, biomarkers, and/or cells may bedetected or isolated by immunofluorescence, immunohistochemistry (IHC),microscopy, fluorescence activated cell sorting (FACS), massspectrometry (MS), mass cytometry (CyTOF), RNA-seq, single cell RNA-seq(described further herein), quantitative RT-PCR, single cell qPCR,Fluorescence In Situ Hybridization (FISH), RNA-FISH, MERFISH (multiplex(in situ) RNA FISH) and/or by in situ hybridization. Other methodsincluding absorbance assays and colorimetric assays are known in the artand may be used herein. detection may comprise primers and/or probes orfluorescently bar-coded oligonucleotide probes for hybridization to RNA(see e.g., Geiss GK, et al., Direct multiplexed measurement of geneexpression with color-coded probe pairs. Nat Biotechnol. 2008Mar;26(3):317-25).

Detection of SLC34A2

In certain embodiments, detection of increased expression of SLC34A2and/or covarying genes indicates that a tumor is sensitive to inhibitionof XPR1:KIDINS220-mediated phosphate export. SLC34A2 expression can bedetermined by detection of the protein or RNA transcripts using anymethod described further herein. Antibodies capable of detecting SLC34A2have been developed (see, e.g., MX35: Yin BW, et al., Monoclonalantibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in humancarcinomas. Cancer Immun. 2008;8:3; Levan K, et al., Immunohistochemicalevaluation of epithelial ovarian carcinomas identifies three differentexpression patterns of the MX35 antigen, NaPi2b. BMC Cancer.2017;17(1):303; and RebMab200: Lopes dos Santos, et al., Rebmab200, aHumanized Monoclonal Antibody Targeting the Sodium Phosphate TransporterNaPi2b Displays Strong Immune Mediated Cytotoxicity against Cancer: ANovel Reagent for Targeted Antibody Therapy of Cancer. PLoS One. 2013;8(7): e70332) and are applicable to the present invention. Detection ofSLC34A2 with an anti-NaPi2b antibody has been described for determiningwhether a cancer is responsive to a NaPi2b-targeted antibody drugconjugate (see, US 2019/0160181 A1). Any future antibodies developed arealso applicable to the present invention. In certain embodiments,antibodies as described in the therapeutic methods may be used.

In certain embodiments, detecting comprises one or more ofimmunohistochemistry (IHC), in situ RNA-seq (Ke, R. et al. In situsequencing for RNA analysis in preserved tissue and cells. Nat. Methods10, 857-860 (2013)), quantitative PCR, RNA-seq, CITE-seq (Stoeckius, M.et al. Simultaneous epitope and transcriptome measurement in singlecells. Nat. Methods 14, 865-868 (2017)), western blot, Fluorescence InSitu Hybridization (FISH), MERFISH (Chen, K. H., Boettiger, A. N.,Moffitt, J. R., Wang, S. & Zhuang, X. Spatially resolved, highlymultiplexed RNA profiling in single cells. Science 348, (2015)),RNA-FISH, mass spectrometry, or FACS.

Copy Number Variation

In certain embodiments, copy number variations (CNV) are detected in atumor (e.g., XPR1) (see, e.g., Carter SL, et al., Absolutequantification of somatic DNA alterations in human cancer. NatBiotechnol. 2012 May; 30(5):413-21; Tirosh, I. et al. Dissecting themulticellular ecosystem of metastatic melanoma by single-cell RNA-seq.Science 352, 189-196 (2016); Sathirapongsasuti, J. F. et al. Exomesequencing-based copy-number variation and loss of heterozygositydetection: ExomeCNV. Bioinformatics 27, 2648-2654 (2011); Krumm, N. etal. Copy number variation detection and genotyping from exome sequencedata. Genome Res. 22, 1525-1532 (2012); de Araújo Lima, L. & Wang, K.PennCNV in whole-genome sequencing data. BMC Bioinformatics 18, 383(2017); Fan, J. et al. Linking transcriptional and genetic tumorheterogeneity through allele analysis of single-cell RNA-seq data.Genome Res. 28, 1217-1227 (2018); Campbell, K. R. et al. Clonealign:statistical integration of independent single-cell RNA and DNAsequencing data from human cancers. Genome Biol. 20, 54 (2019); Chen,M., Gunel, M. & Zhao, H. SomatiCA: Identifying, characterizing andquantifying somatic copy number aberrations from cancer genomesequencing data. PLoS ONE 8, e78143 (2013); Serin Harmanci, A., et al.,CaSpER identifies and visualizes CNV events by integrative analysis ofsingle-cell or bulk RNA-sequencing data. Nat Commun 11, 89 (2020); andOh, et al., Reliable Analysis of Clinical Tumor-Only Whole-ExomeSequencing Data. JCO Clin Cancer Inform. 2020; 4). In certainembodiments, amplifications are detected in XPR1 to identify tumors thatare sensitive to inhibition of XPR1:KIDINS220-mediated phosphate export.In certain embodiments, FISH is used to detect CNVs. In certainembodiments, CNVs are detected by whole-exome sequencing (WES) ortargeted panel sequencing. In certain embodiments, CNVs are detected byinference from a target sequencing panel. In certain embodiments, CNVsare determined using RNA-seq.

Microscopy

In certain embodiments, the morphological changes are detected bymicroscopy. Microscopy is the technical field of using microscopes toview objects and areas of objects that cannot be seen with the naked eye(objects that are not within the resolution range of the normal eye)(see, e.g., Mualla et al., editors. In: Medical Imaging Systems: AnIntroductory Guide [Internet]. Cham (CH): Springer; 2018. Chapter 5.2018 Aug 3. DOI: 10.1007/978-3-319-96520-8_5). Any method of microscopymay be used in the present invention (e.g., optical, electron, andscanning probe microscopy, or X-ray microscopy). In preferredembodiments, phase contrast, fluorescence or confocal microscopy isused.

MS Methods

Biomarker detection may also be evaluated using mass spectrometrymethods. A variety of configurations of mass spectrometers can be usedto detect biomarker values. Several types of mass spectrometers areavailable or can be produced with various configurations. In general, amass spectrometer has the following major components: a sample inlet, anion source, a mass analyzer, a detector, a vacuum system, andinstrument-control system, and a data system. Difference in the sampleinlet, ion source, and mass analyzer generally define the type ofinstrument and its capabilities. For example, an inlet can be acapillary-column liquid chromatography source or can be a direct probeor stage such as used in matrix-assisted laser desorption. Common ionsources are, for example, electrospray, including nanospray andmicrospray or matrix-assisted laser desorption. Common mass analyzersinclude a quadrupole mass filter, ion trap mass analyzer andtime-of-flight mass analyzer. Additional mass spectrometry methods arewell known in the art (see Burlingame et al., Anal. Chem. 70:647 R-716R(1998); Kinter and Sherman, New York (2000)).

Protein biomarkers and biomarker values can be detected and measured byany of the following: electrospray ionization mass spectrometry(ESI-MS), ESI-MS/MS, ESI-MS/(MS)n, matrix-assisted laser desorptionionization time-of-flight mass spectrometry (MALDI-TOF-MS),surface-enhanced laser desorption/ionization time-of-flight massspectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS),secondary ion mass spectrometry (SIMS), quadrupole time-of-flight(Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflexIII TOF/TOF, atmospheric pressure chemical ionization mass spectrometry(APCI-MS), APCI-MS/MS, APCI-(MS).sup.N, atmospheric pressurephotoionization mass spectrometry (APPI-MS), APPI-MS/MS, andAPPI-(MS).sup.N, quadrupole mass spectrometry, Fourier transform massspectrometry (FTMS), quantitative mass spectrometry, and ion trap massspectrometry.

Sample preparation strategies are used to label and enrich samplesbefore mass spectroscopic characterization of protein biomarkers anddetermination biomarker values. Labeling methods include but are notlimited to isobaric tag for relative and absolute quantitation (iTRAQ)and stable isotope labeling with amino acids in cell culture (SILAC).Capture reagents used to selectively enrich samples for candidatebiomarker proteins prior to mass spectroscopic analysis include but arenot limited to aptamers, antibodies, nucleic acid probes, chimeras,small molecules, an F(ab′)₂ fragment, a single chain antibody fragment,an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, aligand-binding receptor, affybodies, nanobodies, ankyrins, domainantibodies, alternative antibody scaffolds (e.g. diabodies etc)imprinted polymers, avimers, peptidomimetics, peptoids, peptide nucleicacids, threose nucleic acid, a hormone receptor, a cytokine receptor,and synthetic receptors, and modifications and fragments of these.

Immunoassays

Immunoassay methods are based on the reaction of an antibody to itscorresponding target or analyte and can detect the analyte in a sampledepending on the specific assay format. To improve specificity andsensitivity of an assay method based on immunoreactivity, monoclonalantibodies are often used because of their specific epitope recognition.Polyclonal antibodies have also been successfully used in variousimmunoassays because of their increased affinity for the target ascompared to monoclonal antibodies Immunoassays have been designed foruse with a wide range of biological sample matrices Immunoassay formatshave been designed to provide qualitative, semi-quantitative, andquantitative results.

Quantitative results may be generated through the use of a standardcurve created with known concentrations of the specific analyte to bedetected. The response or signal from an unknown sample is plotted ontothe standard curve, and a quantity or value corresponding to the targetin the unknown sample is established.

Numerous immunoassay formats have been designed. ELISA or EIA can bequantitative for the detection of an analyte/biomarker. This methodrelies on attachment of a label to either the analyte or the antibodyand the label component includes, either directly or indirectly, anenzyme. ELISA tests may be formatted for direct, indirect, competitive,or sandwich detection of the analyte. Other methods rely on labels suchas, for example, radioisotopes (I¹²⁵) or fluorescence. Additionaltechniques include, for example, agglutination, nephelometry,turbidimetry, Western blot, immunoprecipitation, immunocytochemistry,immunohistochemistry, flow cytometry, Luminex assay, and others (seeImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor& Francis, Ltd., 2005 edition).

Exemplary assay formats include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay, fluorescent, chemiluminescence, andfluorescence resonance energy transfer (FRET) or time resolved-FRET(TR-FRET) immunoassays. Examples of procedures for detecting biomarkersinclude biomarker immunoprecipitation followed by quantitative methodsthat allow size and peptide level discrimination, such as gelelectrophoresis, capillary electrophoresis, planarelectrochromatography, and the like.

Methods of detecting and/or quantifying a detectable label or signalgenerating material depend on the nature of the label. The products ofreactions catalyzed by appropriate enzymes (where the detectable labelis an enzyme; see above) can be, without limitation, fluorescent,luminescent, or radioactive or they may absorb visible or ultravioletlight. Examples of detectors suitable for detecting such detectablelabels include, without limitation, x-ray film, radioactivity counters,scintillation counters, spectrophotometers, colorimeters, fluorometers,luminometers, and densitometers.

Any of the methods for detection can be performed in any format thatallows for any suitable preparation, processing, and analysis of thereactions. This can be, for example, in multi-well assay plates (e.g.,96 wells or 384 wells) or using any suitable array or microarray. Stocksolutions for various agents can be made manually or robotically, andall subsequent pipetting, diluting, mixing, distribution, washing,incubating, sample readout, data collection and analysis can be donerobotically using commercially available analysis software, robotics,and detection instrumentation capable of detecting a detectable label.

Hybridization Assays

Such applications are hybridization assays in which a nucleic acid thatdisplays “probe” nucleic acids for each of the genes to beassayed/profiled in the profile to be generated is employed. In theseassays, a sample of target nucleic acids is first prepared from theinitial nucleic acid sample being assayed, where preparation may includelabeling of the target nucleic acids with a label, e.g., a member of asignal producing system. Following target nucleic acid samplepreparation, the sample is contacted with the array under hybridizationconditions, whereby complexes are formed between target nucleic acidsthat are complementary to probe sequences attached to the array surface.The presence of hybridized complexes is then detected, eitherqualitatively or quantitatively. Specific hybridization technology whichmay be practiced to generate the expression profiles employed in thesubject methods includes the technology described in U.S. Pat. Nos.5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806;5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028;5,800,992; the disclosures of which are herein incorporated byreference; as well as WO 95/21265; WO 96/31622; WO 97/10365; WO97/27317; EP 373 203; and EP 785 280. In these methods, an array of“probe” nucleic acids that includes a probe for each of the biomarkerswhose expression is being assayed is contacted with target nucleic acidsas described above. Contact is carried out under hybridizationconditions, e.g., stringent hybridization conditions as described above,and unbound nucleic acid is then removed. The resultant pattern ofhybridized nucleic acids provides information regarding expression foreach of the biomarkers that have been probed, where the expressioninformation is in terms of whether or not the gene is expressed and,typically, at what level, where the expression data, i.e., expressionprofile, may be both qualitative and quantitative.

Optimal hybridization conditions will depend on the length (e.g.,oligomer vs. polynucleotide greater than 200 bases) and type (e.g., RNA,DNA, PNA) of labeled probe and immobilized polynucleotide oroligonucleotide. General parameters for specific (i.e., stringent)hybridization conditions for nucleic acids are described in Sambrook etal., supra, and in Ausubel et al., “Current Protocols in MolecularBiology”, Greene Publishing and Wiley-interscience, NY (1987), which isincorporated in its entirety for all purposes. When the cDNA microarraysare used, typical hybridization conditions are hybridization in 5xSSCplus 0.2% SDS at 65C for 4 hours followed by washes at 25° C. in lowstringency wash buffer (1xSSC plus 0.2% SDS) followed by 10 minutes at25° C. in high stringency wash buffer (0.1SSC plus 0.2% SDS) (see Shenaet al., Proc. Natl. Acad. Sci. USA, Vol. 93, p. 10614 (1996)). Usefulhybridization conditions are also provided in, e.g., Tijessen,Hybridization With Nucleic Acid Probes″, Elsevier Science PublishersB.V. (1993) and Kricka, “Nonisotopic DNA Probe Techniques”, AcademicPress, San Diego, Calif. (1992).

Sequencing and Nucleic Acid Profiling

In certain embodiments, the invention involves targeted nucleic acidprofiling (e.g., sequencing, quantitative reverse transcriptionpolymerase chain reaction, and the like) (see e.g., Geiss GK, et al.,Direct multiplexed measurement of gene expression with color-coded probepairs. Nat Biotechnol. 2008 Mar;26(3):317-25). In certain embodiments, atarget nucleic acid molecule (e.g., RNA molecule), may be sequenced byany method known in the art, for example, methods of high-throughputsequencing, also known as next generation sequencing or deep sequencing.A nucleic acid target molecule labeled with a barcode (for example, anorigin-specific barcode) can be sequenced with the barcode to produce asingle read and/or contig containing the sequence, or portions thereof,of both the target molecule and the barcode. Exemplary next generationsequencing technologies include, for example, Illumina sequencing, IonTorrent sequencing, 454 sequencing, SOLiD sequencing, and nanoporesequencing amongst others.

In certain embodiments, the invention involves single cell RNAsequencing to detect or quantitate cells that are vulnerable toXPR1:KIDINS220-mediated phosphate export (see, e.g., Kalisky, T.,Blainey, P. & Quake, S. R. Genomic Analysis at the Single-Cell Level.Annual review of genetics 45, 431-445, (2011); Kalisky, T. & Quake, S.R. Single-cell genomics. Nature Methods 8, 311-314 (2011); Islam, S. etal. Characterization of the single-cell transcriptional landscape byhighly multiplex RNA-seq. Genome Research, (2011); Tang, F. et al.RNA-Seq analysis to capture the transcriptome landscape of a singlecell. Nature Protocols 5, 516-535, (2010); Tang, F. et al. mRNA-Seqwhole-transcriptome analysis of a single cell. Nature Methods 6,377-382, (2009); Ramskold, D. et al. Full-length mRNA-Seq fromsingle-cell levels of RNA and individual circulating tumor cells. NatureBiotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F.,Sher, N. & Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed LinearAmplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p666-673,2012).

In certain embodiments, the invention involves plate based single cellRNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-lengthRNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181,doi:10.1038/nprot.2014.006).

In certain embodiments, the invention involves high-throughputsingle-cell RNA-seq. In this regard reference is made to Macosko et al.,2015, “Highly Parallel Genome-wide Expression Profiling of IndividualCells Using Nanoliter Droplets” Cell 161, 1202-1214; Internationalpatent application number PCT/US2015/049178, published as WO2016/040476on Mar. 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-CellTranscriptomics Applied to Embryonic Stem Cells” Cell 161, 1187-1201;International patent application number PCT/US2016/027734, published asWO2016168584A1 on Oct. 20, 2016; Zheng, et al., 2016, “Haplotypinggermline and cancer genomes with high-throughput linked-read sequencing”Nature Biotechnology 34, 303-311; Zheng, et al., 2017, “Massivelyparallel digital transcriptional profiling of single cells” Nat. Commun.8, 14049 doi: 10.1038/ncomms14049; International patent publicationnumber WO2014210353A2; Zilionis, et al., 2017, “Single-cell barcodingand sequencing using droplet microfluidics” Nat Protoc. Jan;12(1):44-73;Cao et al., 2017, “Comprehensive single cell transcriptional profilingof a multicellular organism by combinatorial indexing” bioRxiv preprintfirst posted online Feb. 2, 2017, doi: dx.doi.org/10.1101/104844;Rosenberg et al., 2017, “Scaling single cell transcriptomics throughsplit pool barcoding” bioRxiv preprint first posted online Feb. 2, 2017,doi: dx.doi.org/10.1101/105163; Rosenberg et al., “Single-cell profilingof the developing mouse brain and spinal cord with split-pool barcoding”Science 15 Mar. 2018; Vitak, et al., “Sequencing thousands ofsingle-cell genomes with combinatorial indexing” Nature Methods,14(3):302-308, 2017; Cao, et al., Comprehensive single-celltranscriptional profiling of a multicellular organism. Science,357(6352):661-667, 2017; Gierahn et al., “Seq-Well: portable, low-costRNA sequencing of single cells at high throughput” Nature Methods 14,395-398 (2017); and Hughes, et al., “Highly Efficient,Massively-Parallel Single-Cell RNA-Seq Reveals Cellular States andMolecular Features of Human Skin Pathology” bioRxiv 689273; doi:doi.org/10.1101/689273, all the contents and disclosure of each of whichare herein incorporated by reference in their entirety.

In certain embodiments, the invention involves single nucleus RNAsequencing. In this regard reference is made to Swiech et al., 2014, “Invivo interrogation of gene function in the mammalian brain usingCRISPR-Cas9” Nature Biotechnology Vol. 33, pp. 102-106; Habib et al.,2016, “Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adultnewborn neurons” Science, Vol. 353, Issue 6302, pp. 925-928; Habib etal., 2017, “Massively parallel single-nucleus RNA-seq with DroNc-seq”Nat Methods. 2017 Oct;14(10):955-958; International patent applicationnumber PCT/US2016/059239, published as WO2017164936 on Sep. 28, 2017;International patent application number PCT/US2018/060860, published asWO/2019/094984 on May 16, 2019; International patent application numberPCT/US2019/055894, published as WO/2020/077236 on Apr. 16, 2020; andDrokhlyansky, et al., “The enteric nervous system of the human and mousecolon at a single-cell resolution,” bioRxiv 746743; doi:doi.org/10.1101/746743, which are herein incorporated by reference intheir entirety.

In certain embodiments, the invention involves the Assay for TransposaseAccessible Chromatin using sequencing (ATAC-seq) as described. (See,e.g., Buenrostro, et al., Transposition of native chromatin for fast andsensitive epigenomic profiling of open chromatin, DNA-binding proteinsand nucleosome position. Nature methods 2013; 10 (12): 1213-1218;Buenrostro et al., Single-cell chromatin accessibility revealsprinciples of regulatory variation. Nature 523, 486-490 (2015);Cusanovich, D. A., Daza, R., Adey, A., Pliner, H., Christiansen, L.,Gunderson, K. L., Steemers, F. J., Trapnell, C. & Shendure, J. Multiplexsingle-cell profiling of chromatin accessibility by combinatorialcellular indexing. Science. 2015 May 22;348(6237):910-4. doi:10.1126/science.aab1601. Epub 2015 May 7; US20160208323A1;US20160060691A1; and WO2017156336A1).

Screening Methods

In certain embodiments, therapeutic agents that are capable ofinhibiting XPR1:KIDINS220-mediated phosphate export are screened.Screening may be performed in vitro or in vivo. For example, agents thatmodulate phosphate export in a tumor microenvironment may be screened invivo. In certain embodiments, cancer cell lines can be assayed forphosphate efflux as described herein.

A further aspect of the invention relates to a method for identifying anagent capable of inhibiting XPR1:KIDINS220-mediated phosphate export,comprising: a) applying a candidate agent to a cancer cell or cellpopulation; b) detecting modulation of phosphate efflux in the cell orcell population by the candidate agent, thereby identifying the agent.The term “modulate” broadly denotes a qualitative and/or quantitativealteration, change or variation in that which is being modulated. Wheremodulation can be assessed quantitatively — for example, wheremodulation comprises or consists of a change in a quantifiable variablesuch as a quantifiable property of a cell or where a quantifiablevariable provides a suitable surrogate for the modulation — modulationspecifically encompasses both increase (e.g., activation) or decrease(e.g., inhibition) in the measured variable. The term encompasses anyextent of such modulation, e.g., any extent of such increase ordecrease, and may more particularly refer to statistically significantincrease or decrease in the measured variable. By means of example,modulation may encompass an increase in the value of the measuredvariable by at least about 10%, e.g., by at least about 20%, preferablyby at least about 30%, e.g., by at least about 40%, more preferably byat least about 50%, e.g., by at least about 75%, even more preferably byat least about 100%, e.g., by at least about 150%, 200%, 250%, 300%,400% or by at least about 500%, compared to a reference situationwithout said modulation; or modulation may encompass a decrease orreduction in the value of the measured variable by at least about 10%,e.g., by at least about 20%, by at least about 30%, e.g., by at leastabout 40%, by at least about 50%, e.g., by at least about 60%, by atleast about 70%, e.g., by at least about 80%, by at least about 90%,e.g., by at least about 95%, such as by at least about 96%, 97%, 98%,99% or even by 100%, compared to a reference situation without saidmodulation. Preferably, modulation may be specific or selective, hence,one or more desired phenotypic aspects of an immune cell or immune cellpopulation may be modulated without substantially altering other(unintended, undesired) phenotypic aspect(s).

The term “agent” broadly encompasses any condition, substance or agentcapable of modulating one or more phenotypic aspects of a cell or cellpopulation as disclosed herein. Such conditions, substances or agentsmay be of physical, chemical, biochemical and/or biological nature. Theterm “candidate agent” refers to any condition, substance or agent thatis being examined for the ability to modulate one or more phenotypicaspects of a cell or cell population as disclosed herein in a methodcomprising applying the candidate agent to the cell or cell population(e.g., exposing the cell or cell population to the candidate agent orcontacting the cell or cell population with the candidate agent) andobserving whether the desired modulation takes place.

Agents may include any potential class of biologically activeconditions, substances or agents, such as for instance antibodies,proteins, peptides, nucleic acids, oligonucleotides, small molecules, orcombinations thereof, as described herein.

The screening methods can be utilized for evaluating environmentalstress and/or state, for screening of chemical libraries, and to screenor identify structural, syntenic, genomic, and/or organism and speciesvariations. For example, a culture of cells, can be exposed to anenvironmental stress, such as but not limited to heat shock, osmolarity,hypoxia, cold, oxidative stress, radiation, starvation, a chemical (forexample a therapeutic agent or potential therapeutic agent) and thelike. After the stress is applied, a representative sample can besubjected to analysis, for example at various time points, and comparedto a control, such as a sample from an organism or cell, for example acell from an organism, or a standard value. By exposing cells, orfractions thereof, tissues, or even whole animals, to different membersof the chemical libraries, and performing the methods described herein,different members of a chemical library can be screened for their effecton phenotypes thereof simultaneously in a relatively short amount oftime, for example using a high throughput method.

In some embodiments, screening of test agents involves testing acombinatorial library containing a large number of potential modulatoragents. A combinatorial chemical library may be a collection of diversechemical agents generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library, such asa polypeptide library, is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given agent length (forexample the number of amino acids in a polypeptide agent). Millions ofchemical agents can be synthesized through such combinatorial mixing ofchemical building blocks.

In certain embodiments, cancers sensitive to inhibition ofXPR1:KIDINS220-mediated phosphate export are identified by screeningtumor cells from a cancer. The method may comprise applying an inhibitorof XPR1:KIDINS220-mediated phosphate export to a cancer cell or cellpopulation (e.g., RBD); and detecting the phosphate concentration in thecell or cell population, wherein the cancer is sensitive if thephosphate concentration is increased as compared to a control cell orpopulation not treated with the inhibitor. In certain embodiments, theinhibitor of XPR1:KIDINS220-mediated phosphate export is one or moretherapeutic agents according to any embodiment herein. In certainembodiments, the cancer cell or population is obtained or derived from asubject in need thereof. For example, the method can includepersonalized therapeutics, such that tumor cells from a subject aregrown and assayed for sensitivity to inhibition ofXPR1:KIDINS220-mediated phosphate export. In certain embodiments, celllines derived from specific cancers are screened.

KITS OR PHARMACEUTICAL SYSTEMS

The present compositions may be assembled into kits or pharmaceuticalsystems for use in ameliorating a neoplasia. Kits or pharmaceuticalsystems according to this aspect of the invention comprise a carriermeans, such as a box, carton, tube or the like, having in closeconfinement therein one or more container means, such as vials, tubes,ampoules, or bottles. The kits or pharmaceutical systems of theinvention may also comprise associated instructions for using the agentsof the invention. The present invention also may comprise a kit with adetection reagent that binds to one or more biomarkers or can be used todetect one or more biomarkers.

Further embodiments are illustrated in the following Examples which aregiven for illustrative purposes only and are not intended to limit thescope of the invention.

EXAMPLES Example 1 - Phosphate Dysregulation via the NovelXPR1:KIDINS220 Protein Complex is a Therapeutic Vulnerability in OvarianCancer

Applicants hypothesized that phosphate dysregulation had therapeuticpotential for cancer by examining the cancer Dependency Map for targetswith a viability defect in ovarian cancer ^(18,19). Across all 733cancer cell lines screened with CRISPR/Cas9, the phosphate exporter XPR1has one of the most selective and predictive profiles across all genes(FIG. 1A), and especially for the smaller number of genetic dependenciesobserved in ovarian and uterine cancers. XPR1 inactivation has no effecton most cancer cell lines while ovarian and uterine cancer cell linesdisplay a high degree of dependency on XPR1 (15 out of 63 cell lineswith a CERES score < -0.6). This selective dependency profile iscomparable to emerging (e.g. WRN ²⁰, EGLN1 ²¹) and well-establishedcancer therapeutic targets (e.g., EGFR or IKZF1). Therapeutic inhibitionof XPR1 is also more feasible¹² than other highly selective yetchallenging targets (e.g., transcription factors such as PAX8, HGM1, andWT1).

Applicants next pursued the molecular basis of the selective dependencyon XPR1. Applicants built multivariate predictive models from thefeatures most correlated with XPR1 dependency among the many cellularand molecular features of each cancer cell line ²². Using variableimportance analysis of the most accurate model (r = 0.391) (FIG. 1A,Y-axis), the most predominant predictive feature is expression of thephosphate importer gene SLC34A2 (FIG. 1B, 0.38 normalized Giniimportance). The correlation between high SLC34A2 expression and XPR1dependency is mostly driven by a strong relationship in ovarian anduterine tissues (R² = 0.15, n = 731 in all versus R² = 0.33, n = 63 inovarian/uterine, FIG. 2A). Interestingly, although increased SLC34A2expression in ovarian cancer is of unknown consequence, its enhancedexpression is well established ²³⁻²⁵.

The observation that high expression of SLC34A2 is associated withdependency on XPR1 led us to hypothesize that accumulation ofintracellular phosphate upon XPR1 inactivation is selectively toxic toovarian and uterine cancer cells (FIG. 1C). Applicants first validatedthese observations by assessing cellular viability after geneticinactivation of XPR1 in a panel of ovarian and uterine cancer cell lineswith low or high SLC334A2 expression. XPR1 inactivation by CRISPR/Cas9decreased cellular viability in SLC34A2-high cell lines to a levelsimilar to inactivation of pan-essential controls. In contrast,Applicants found no decrease of viability in SLC34A2-low cell lines(FIG. 1D). Similarly, this viability defect is seen when XPR1 issuppressed using shRNA reagents (FIGS. 2B-D).

Applicants next sought to systematically determine the extent to whichSLC34A2 —or other genes — is necessary to confer dependency on XPR1 byconducting a genome-scale genetic loss-of-function modifier screen(FIGS. 3A-B ²⁶). Of the ~17,000 genes tested, the only genetic knockoutwhich rescued the viability defects of XPR1 inactivation was SLC34A2(FIG. 1E, FIG. 3C). Applicants further tested the necessity andsufficiency of SLC34A2 by stably inactivating or overexpressing SLC34A2in several cell lines. Altered SLC34A2 expression alone caused no growthor viability defects (see the dependency profile in FIG. 1B), but itsexpression is both necessary and sufficient to confer XPR1 dependency(FIG. 1F).

Dependencies sensitive to the metabolic environment can often bedifferent between tissue culture and more physiologically relevantsettings. Applicants next investigated if Applicants could find evidenceof phosphate dysregulation in primary ovarian or uterine tumors and ifApplicants could identify tumor samples where XPR1 might be predicted tobe a dependency. Applicants first evaluated the relationship betweenXPR1 and SLC34A2, Applicants first sought evidence in primary samplesfrom tumors in the The Cancer Genome Atlas (TCGA)^(27,28) and normaltissue from the Genotype-Tissue Expression project (GTEx) ^(29,30).Applicants found that ovarian and uterine cancers are among the fewtissues which display enhanced SLC34A2 expression (FIG. 5A).Interestingly, although the fallopian tube does not have a known role inphosphate homeostasis, these normal samples have slightly higherexpression of SLC34A2 relative to normal ovarian or uterine tissue (FIG.4A). Compared to normal fallopian tube epithelium, which may be a cellof origin for these tumors (Köbel et al. 2008; Piek et al. 2001; Jarboeet al. 2008; Hu et al. 2020), there was a 16.3- and 2.66-fold increasein ovarian and uterine cancers, respectively (FIG. 4A). Most tumorsamples had equivalent SLC34A2 expression as cancer cell lines highlydependent on XPR1.

Applicants next sought evidence of what is driving the high levels ofSLC34A2 expression in these tumor samples. SLC34A2 has previously beenreported to be a component of the paired box 8 (PAX8) transcriptionalprogram ³⁵. PAX8 is a lineage-defining transcription factor which isamplified and upregulated in the course of ovarian carcinogenesis(Mittag et al. 2007; Cheung et al. 2011). Applicants found that all theovarian cell lines expressing high levels of SLC34A2 also express highlevels of PAX8 (FIGS. 5B-C). This may indicate that PAX8 expressiondrives the over-expression of SLC34A2 in ovarian cancer, an appealinghypothesis that requires further study.

According to the therapeutic hypothesis (FIG. 1C), increased expressionof SLC34A2 in tumor samples would create an increased demand forphosphate efflux, and a reliance on XPR1. In line with this hypothesis,Applicants found strong evidence for copy number amplifications of XPR1in ovarian and uterine cancer (FIG. 4B) ²⁷. The XPR1 locus undergoessignificant and recurrent copy gains or amplifications in ovarian cancer(FIGS. 4C; 4G = 0.0015 by GISTIC ³⁸), that are often focal and onlyinclude XPR1. Uterine samples also had frequent XPR1 copy gains (44% ofsamples), although they were not significantly recurrent (FIG. 5D,q=0.568; ref. ³⁹). Accordingly, XPR1 mRNA expression levels correlatewith XPR1 copy number alterations, but other mechanisms are additionallydriving XPR1 mRNA expression (FIG. 4C). While the causative event behindincreased expression of both XPR1 and SLC34A2 is not clear from theseanalyses, Applicants propose that reliance upon increased XPR1expression is a consequence of increased SLC34A2. Together, these dataindicate that ovarian tumors have altered phosphate homeostasis relativeto normal tissue, and that high expression of SLC34A2 may serve as atherapeutic biomarker to predict response to XPR1 inhibition.

Applicants next sought evidence that the XPR1 dependency is not anartifact of supra-physiological phosphate levels from in vitro culture.Applicants first noted that while phosphate concentrations varydrastically between the growth medium of different cell lines (rangingfrom 15 to nearly 80 mg/dL), there was no correlation with the strengthof the XPR1 dependency (R² = 0.00, n = 658, FIG. 6A). Furthermore, whenApplicants lowered the phosphate concentration in a highly XPR1dependent cell line by ~10-fold (from 72.8 to 7.8 mg/dL), the cellsremained highly dependent on XPR1 (FIGS. 6B-C), indicating thatphysiological concentrations of extracellular phosphate are sufficientto inhibit cancer cell survival.

Applicants next directly evaluated whether XPR1 inactivation wouldaffect the initiation and maintenance of cancer cell line xenografts.Applicants first developed in a CRISPR/Cas9-based tumor formationcompetition assay, in which a small library of sgRNA was delivered vialentivirus to cancer cell lines in vitro and rapidly inoculatedsubcutaneously (FIG. 7A),. XPR1 inactivation sgRNAs were depleted causeda competitive disadvantage in this assay, and its depletion was noted inboth tissue culture and all xenograft tumors in the two SLC34A2high-expressing cell lines tested (FIGS. 4D-E, Supplemental table 1 andFIGS. 7BE). In contrast, other metabolic genes, most notably theferroptosis regulator GPX4, showed differences between their in vitroand in vivo viability effects (FIGS. 4D-E), indicating this assay candetect dependencies which are sensitive to the metabolic environment.Next, Applicants evaluated the effect of XPR1 suppression on establishedovarian peritoneal carcinomatosis, a clinically relevant model.Applicants injected a SLC34A2 high-expressing ovarian cancer cell lineexpressing luciferase and a doxycycline-inducible shRNA targeting XPR1or a seed-matched control shRNA (FIGS. 2 ) into the peritoneal cavity(see FIG. 8A and methods for study design and timeline). After threeweeks of tumor growth and acclimation to physiologically relevant levelsof phosphate, tumor burden was consistent across animals (FIGS. 8B-D),and so Applicants treated the animals with control or doxycycline diets(FIGS. 4F-G, inset, n = 4 per group). XPR1 suppression significantlydelayed tumor progression for two weeks following doxycycline treatment(FIG. 4F), whereas induction of the control shRNA had no significanteffect on tumor growth (FIG. 4G). Taken together, the results of thesubcutaneous sgRNA competition assays and the intraperitoneal xenograftsindicate that the XPR1 dependency is retained in vivo with physiologicallevels of inorganic phosphate.

Applicants next pursued a mechanistic understanding of whySLC34A2-overexpressing cell lines lose viability after XPR1 knockout.Consistent with the therapeutic hypothesis, Applicants observed a 2-4fold increase in intracellular phosphate after knockdown of XPR1 whichoccurred in the same timeframe as viability defects (FIG. 9A, FIG. 10A).As the mechanisms to manage intracellular phosphate are notwell-understood in human biology¹⁰, Applicants pursued gene expressionprofiling. Applicants used MixSeq⁴⁰, a multiplexed single-cell RNAsequencing assay to compare the transcriptional response across celllines with varying degrees of dependency on XPR1.Applicants collecteddata from 2,501 single cells, representing 8 different ovarian anduterine cancer cell lines, 4 days after XPR1 inactivation usingCRISPR/Cas9. This time-point was chosen to identify primary phosphatehomeostatic mechanisms rather than secondary effects. At this time-pointthere were no significant differences in the number of cells betweencontrol and XPR1 inactivation conditions, indicating viability effectshad not yet occurred.

Applicants observed a strong and highly correlated transcriptionalresponse among the most XPR1-dependent cell lines. While there are fewcanonical gene sets represented in this signature (FIGS. 10 f-i ) thistranscriptional program — at least in part — attempts to restorephosphate homeostasis which Applicants conclude based on thedifferential expression of several genes. First, Applicants observed theup-regulation of FGF23 (FIG. 9F), a critical phosphate homeostatichormone typically expressed by osteogenic bone cells in response toelevated serum phosphate ⁴¹. This is consistent with the increasedintracellular phosphate Applicants observe (FIG. 9A). Fallopian tube,ovarian, and uterine tissues have not been implicated in phosphatehomeostasis, and so these cell lines’ ability — though partial (FIG.10I) — to regulate FGF23 is surprising.⁹

Applicants also observed the downregulation of phosphate import. Twophosphate importer genes, SLC20A1 and SLC34A2, were significantlydecreased after XPR1 knockout (FIG. 9F). SLC34A2 protein levels are alsodramatically reduced after knockout of XPR1 (FIG. 9 ), and XPR1 knockoutcauses a ~60% decrease in phosphate uptake, which is most likely due tothe partial suppression of SLC34A2 (FIG. 9 ). These results are somewhatparadoxical given that full knockout of SLC34A2 can fully rescue theXPR1 viability defect (compare FIGS. 1E-F) and indicate thedysregulation of SLC34A2. Incomplete suppression of SLC34A2 may be dueto an alternative regulatory mechanism (such as that of PAX8 ³⁵), butthis hypothesis requires further study. Overall, these data highlightthe presence of a phosphate-sensing mechanism which attempts tocounteract increased intracellular phosphate.

These results prompted Applicants to seek direct evidence that phosphateefflux is required for cell survival. Correlated dependencyrelationships often identify genes that are part of the same pathway orprocesses inform the mechanisms of cell killing^(42,43), and highlycorrelated dependencies are often part of the same complex. Applicantswere surprised that the dependency profile of an unrelated gene — KinaseD Interacting Substrate 220⁴⁴⁻⁴⁷ (KIDINS220, FIG. 11 and FIGS. 12A-B) -was strongly correlated with the dependency on XPR1. XPR1 and KIDINS220expression is highly correlated across diverse tissues (FIG. 12C), whichled us to hypothesize that these genes likely co-operate to achievephosphate efflux. Indeed, loss of KIDINS220 or XPR1 causes similarincreases in intracellular phosphate (FIG. 12D). While the exactmechanisms of phosphate efflux is unknown, previous work suggests thatXPR1 and other orthologs use a transmembrane protein domain — called theEXS domain — to move between the golgi apparatus and the plasma membranein order to achieve phosphate eflux move between the plasma membrane andthe golgi apparatus to effect phosphate efflux, and this movementrequires the C-terminal EXS domain^(48,49). Interestingly, KIDINS220also moves between these compartments⁵⁰. This prompted us to evaluatewhether the localization and phosphate efflux of XPR1 requiresKIDINS220.

By using publicly available mass spectrometry databases (FIG. 12E),co-immunoprecipitation, and immunofluorescence, Applicants confirmedthat XPR1 and KIDINS220 form a protein complex required for phosphateefflux. First, Applicants were able to identify the complex usingconventional immunoprecipitation approaches. Applicants found that thehis interaction is not mediated by the N-terminal SPX domain^(17,51),but appears to requires a portion of the C-terminal EXS domain of XPR1,which is critical for proper localization of the protein ⁴⁸ (FIG. 11B,FIGS. 13A-C). Second, Applicants noted that inactivation of KIDINS220causes mislocalization of XPR1 (FIG. 11C). Finally, Applicants directlymeasured phosphate efflux in ovarian cancer cell lines, and found thatinactivation of XPR1 or KIDINS220 decreased phosphate efflux to similardegrees (FIG. 11D). The loss of KIDINS220 protein after XPR1 knockout(FIG. 2B) and the observation that one cannot compensate for loss of theother (FIG. 11A) indicates that these proteins are required for thefunction of the same protein complex and not separate efflux complexes.Thus, the co-dependency of two previously unconnected transmembraneproteins converges on a fundamental inability to export inorganicphosphate.

Applicants confirmed that XPR1 and KIDINS220 form a protein complexrequired for phosphate efflux with several experiments. First, inpublicly available mass spectrometry datasets, XPR1 and KIDINS220interact together, and with other proteins within the secretory pathway(FIG. 13A). Second, Applicants confirmed the XPR1:KIDINS220 proteincomplex with co-immunoprecipitation, and found that the EXS domain ofXPR1 is critical for this interaction (FIG. 11B). Interestingly, theN-terminal SPX domain of XPR1 — which has been implicated in phosphateefflux and regulation — was neither necessary nor sufficient to bindKIDINS220. In line with this physical interaction, KIDINS220 proteinlevels decrease dramatically upon suppression of XPR1 (FIG. 2B). Third,by using immunofluorescence, Applicants noted that KIDINS220inactivation causes XPR1 to mislocalize from puncate secretory vesiclesto a more diffuse cytoplasmic pattern. Finally, Applicants directlymeasured phosphate efflux and found that XPR1 and KIDINS220 inactivationimpaired phosphate efflux to the same degree (FIG. 11D). Importantly,the loss of KIDINS220 protein after XPR1 knockout (FIG. 2B) and theobservation that one cannot compensate for loss of the other (FIG. 11A)indicates that these proteins are required for the function of the sameprotein complex and not separate efflux complexes. Thus, theco-dependency of two previously unconnected transmembrane proteinsconverges on a fundamental inability to export inorganic phosphate.

To further confirm that the phosphate efflux capacity of XPR1 isrequired for cancer cell viability, Applicants employed hypomorphicmutations of XPR1 which have been reported in a rare brain calcificationdisorder ^(15,52). While knockout of endogenous XPR1 can be rescued byectopic expression of full-length, wildtype XPR1, the L218S mutation didnot support cellular viability despite proper localization (FIG. 11 eand FIGS. 13 ). This mutation has some residual phosphate effluxcapacity¹⁵, perhaps indicating these cancer cells are sensitive to evenpartial loss of function. These data clearly indicate that functionalphosphate efflux is required for cellular viability in ovarian cancerwith SLC34A2 overexpression.

Vacuole-like structures have been described as phosphate-storagemechanisms in plants. During the course of these studies Applicantsnoticed that the XPR1-dependent cells formed large, “vacuole-like”structures in the cytoplasm, acytoplasma striking and highly penetrantchange in the morphology of cells with high SLC34A2 expression afterXPR1 or KIDINS220 inactivation (FIG. 11F and FIG. 14A). These cellsformed large, “vacuole-like” structures in the cytoplasm. The structureswere observed in all XPR1-dependent ovarian cancer cell lines tested,invariably preceded loss of cell viability (Supplemental movies), andfrequently appeared in multinucleated cells. In some cases, the entirecytoplasm was filled with these structures which Applicants show are notderived from the Endoplasmic Reticulum, Golgi Apparatus, Mitochondria,Nucleus, or Early Endosomes (FIG. 14B). However, these structures werestained with the acidic dye Lysotracker (FIG. 11G) and appear toco-localize with the lysosomal marker LAMP1 (FIG. 14B), suggesting theymay be related to the lysosomal-system. Ultrastructural analysis bytransmission electron microscopy revealed that these structures arebound by a double-membrane and are often fenestrated (FIG. 11H). They donot have the electron-dense appearance typical of lysosomes, butApplicants did observe the fusion of lysosomes with these “vacuole-like”structures. Applicants hypothesize that these are novel structures butare related to phosphate-storage structures which have been reported inhuman biology ⁵³⁻⁵⁵ or other organisms. Perhaps inactivation of XPR1 orKIDINS220 causes the excessive cytoplasmic accumulation and fusion ofvesicles used in the process of phosphate efflux. ^(49,56,57) Whetherthese structures are supporting cell survival or the ‘cytoplasmiccrowding’ observed in these cancer cell lines is related to the observedcell cycle arrest (FIG. 9C) and loss of cell viability is yet to bedetermined.

Treating cells with high concentrations of phosphate has been shown tobe toxic before ^(58,59), but this study exploits a unique syntheticlethal interaction between SLC34A2 overexpression and XPR1 inhibition.XPR1 inactivation, regardless of high expression of SLC34A2, is nottoxic in tissues with known roles for organismal phosphate homeostasis(e.g., lung cancer ^(60,61), FIG. 2A). This is possibly due to feedbackmechanisms which suppress phosphate import upon increased intracellularphosphate (such as observed in FIGS. 9 and reported before¹⁶). Incontrast, over-expression of SLC34A2 — perhaps through PAX8 or anotherpathway — breaks this feedback mechanism in ovarian and uterine cancers,leading to accumulation of intracellular phosphate and cell death. Theidentity of this feedback mechanism, and whether its inhibition wouldenhance the XPR1 dependency, requires further study.

Therapeutic strategies to exploit phosphate dysregulation in ovariancancer should focus on inhibiting the phosphate efflux capacity ofXPR1:KIDINS220, perhaps through the extracellular binding of proteinligands¹². Applicants expect that a large patient population withSLC34A2 overexpression would respond to such a therapy, althoughintra-tumor heterogeneity would need to be evaluated^(23,25). Theside-effects of inhibiting XPR1:KIDINS220 are likely minimal andmanageable. Although phosphate toxicity is unlikely to be toxic toindividual cells — as most cell lines are not dependent on XPR1 orKIDINS220 — organismal perturbations in phosphate homeostasis should beavoided^(13,41). Nevertheless, transient or partial inhibition of XPR1are likely to be tolerated^(15,52,62) and there is precedent for theclinical management of hypophosphatemia ^(41,63). Applicants hope thatsuch a strategy will one day benefit cancer patients.

Example 2 - Therapeutic Targeting of XPR1:KIDINS220

Applicants show that XRBD is a drug-like inhibitor of XPR1 by inhibitingXPR1-dependent phosphate efflux in ovarian cancer cell lines (FIG. 17A).Using stable knockout of XPR1 or KIDINS220 in 293T cells Applicants showthat XPR1 and KIDINS220 are in a protein complex, and KIDINS220 proteinlevels can be a surrogate marker for XPR1 inactivation and a potentialbiomarker of therapeutic response (FIG. 17B and corresponding to FIG.2B). Using XRBD flow cytometry analysis of the 293T cells analyzed inFIG. 17B Applicants show that KIDINS220 inactivation leads to a drasticdecrease in XPR1 cell-surface localization (FIG. 17C and correspondingto FIG. 11C). Applicants further show that XRBD is a drug-like inhibitorof XPR1 by treating ovarian cancer cell lines with XRBD, such that thetreatment causes viability defects in the ovarian cancer cell lines(FIG. 17D). The XRBD sensitivity (decrease in cellular viability afterfive-day treatment with the top dose of XRBD relative to vehiclecontrol) correlates to each cell lines’ XPR1 inactivation sensitivity(assessed by CRISPR viability assays). Applicants analyzed XRBDtreatment in a small panel of lung cancer cell lines (FIG. 17E andcorresponding to FIG. 5A). Although expressing the predictive biomarkerat high levels (SLC34A2), lung cancer cell lines can suppress theSLC34A2 phosphate importer to protect against cell viability defectsupon XPR1 inhibition. Using glycerol gradient sedimentation analysis ofXPR1-containing native protein complexes with or without KIDINS220inactivation Applicants show that KIDINS220 inactivation leads to adrastic reduction in the molecular weight of XPR1 protein complexes,suggesting altered protein complex composition (FIG. 17F). Using XPR1RT-PCR Applicants show that inactivation of XPR1 leads to a change inphosphate-related genes (e.g., upregulation of FGF23), indicating aphosphate dysregulation state (FIG. 17G).

The sequence of the XRBD protein used in FIGS. 17 is:

MLVMEGSAFSKPLKDKINPWGPLIVMGILVRAGASVQRDSPHQIFNVTWRVTNLMTGQTANATSLLGTMTDTFPKLYFDLCDLVGDYWDDPEPDIGDGCRTPGGRRRTRLYDFYVCPGHTVPIGCGGPGEGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKDQGPCYDSSVSSGVQGATPGGRCNPLVLEFTDAGRKASWDAPKVWGLRLYRSTGADPVTRFSLTRQVLNVGPRVPIGSVDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 2).

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Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

What is claimed is:
 1. A method of treating cancer in a subject in needthereof comprising administering to the subject one or more therapeuticagents capable of inhibiting XPR1:KIDINS220-mediated phosphate export.2. The method of claim 1, wherein the cancer is selected from the groupconsisting of ovarian cancer, uterine cancer, breast cancer, bile ductcancer, liver and lung cancer.
 3. The method of claim 1 or 2, whereinthe cancer is characterized by higher expression of SLC34A2 in tumortissue as compared to expression in normal tissue.
 4. The method of anyof claims 1 to 3, wherein the one or more therapeutic agents inhibit theexpression or activity of XPR1, inhibit the expression or activity ofKIDINS220, and/or disrupt XPR1/KIDINS220 interaction.
 5. The method ofclaim 4, wherein the one or more therapeutic agents comprise a receptorbinding domain (RBD) protein derived from an enveloped virusglycoprotein and capable of interacting with the XPR1 membrane receptor.6. The method of claim 5, wherein the RBD protein is a fusion protein,wherein the fusion protein comprises a domain capable of dimerizationand/or stabilization of the protein.
 7. The method of claim 6, whereinthe RBD protein is fused to an Fc domain, glutathione S-transferase(GST), and/or serum albumin.
 8. The method of any of claims 5 to 7,wherein the RBD protein is derived from xenotropic or polytropic murineleukemia retrovirus (X- and P-MLV) Env.
 9. The method of any of claims 6to 8, wherein the RBD fusion protein comprises the amino acid sequence:MLVMEGSAFSKPLKDKINPWGPLIVMGILVRAGASVQRDSPHQIFNVTWRVTNLMTGQTANATSLLGTMTDTFPKLYFDLCDLVGDYWDDPEPDIGDGCRTPGGRRRTRLYDFYVCPGHTVPIGCGGPGEGYCGKWGCETTGQAYWKPSSSWDLISLKRGNTPKDQGPCYDSSVSSGVQGATPGGRCNPLVLEFTDAGRKASWDAPKVWGLRLYRSTGADPVTRFSLTRQVLNVGPRVPIGSVDVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGK(SEQ ID NO: 2).


10. The method of any of claims 5 to 9, wherein the one or moretherapeutic agents comprise a vector encoding for the RBD protein. 11.The method of claim 4, wherein the one or more therapeutic agentscomprise an antibody specific for XPR1, an antibody specific forKIDINS220, or an antibody specific to the XPR1/KIDINS220 proteincomplex.
 12. The method of claim 11, wherein the antibody targets aWalker A/B motif of KIDINS220.
 13. The method of claim 4, wherein theone or more therapeutic agents comprise a degrader molecule.
 14. Themethod of claim 13, wherein the degrader molecule is a LYTAC molecule,whereby a cell surface protein is targeted.
 15. The method of claim 4,wherein the one or more therapeutic agents comprise a genetic modifyingagent capable of inhibiting the expression of XPR1 or KIDINS220.
 16. Themethod of claim 15, wherein the genetic modifying agent comprises aCRISPR-Cas system, a RNAi, a zinc finger nuclease, a TALE system, or ameganuclease.
 17. The method of claim 16, wherein the CRISPR-Cas systemis a CRISPR-Cas base editing system, a prime editor system, or a CASTsystem.
 18. The method of any of claims 1 to 17, further comprisingadministering to the subject one or more therapeutic agents capable ofinhibiting the expression or activity of FGF23, capable of inhibitingthe suppression of SLC34A2, or capable of modulating one or more genesup or down-regulated in response to XPR1 inhibition.
 19. The method ofany of claims 1 to 18, wherein one or more therapeutic agents capable ofinhibiting XPR1:KIDINS220-mediated phosphate export are co-administeredwithin a standard of care treatment regimen.
 20. The method of claim 19,wherein the standard of care treatment regimen comprises surgery andchemotherapy.
 21. The method of any of claims 19, wherein the standardof care treatment regimen comprises administration of an immunotherapy,checkpoint blockade therapy or a PARP inhibitor.
 22. A method oftreating cancer in a subject in need thereof comprising: detectingtumors sensitive to phosphate dysregulation by detecting increasedexpression of SLC34A2 relative to a control, wherein if the subject hasa tumor sensitive to phosphate dysregulation, including administrationof one or more therapeutic agents capable of inhibitingXPR1:KIDINS220-mediated phosphate export according to any of claims 4 to17; if the subject does not have a tumor sensitive to phosphatedysregulation, administering a standard of care treatment that does notinclude administration of one or more therapeutic agents capable ofinhibiting XPR1:KIDINS220-mediated phosphate export.
 23. The method ofclaim 22, wherein the cancer is selected from the group consisting ofovarian cancer, uterine cancer, breast cancer, bile duct cancer, liverand lung cancer.
 24. The method of claim 22 or 23, wherein the standardof care treatment comprises one or more of surgery, chemotherapy,immunotherapy, checkpoint blockade therapy or administration of a PARPinhibitor.
 25. The method of any one of claims 1 to 24, furthercomprising monitoring the efficacy of the treatment comprising detectingin a tumor sample obtained from the subject the expression of one ormore genes selected from the group consisting of SLC34A2, SLC20A1 andFGF23, wherein the treatment is effective if SLC34A2 and/or SLC20A1 aredecreased, and/or FGF23 is increased.
 26. The method of any one ofclaims 1 to 25, further comprising monitoring the efficacy of thetreatment comprising detecting increased morphological changesassociated with phosphate dysregulation in tumor cells obtained from thesubject, wherein the treatment is effective if increased morphologicalchanges associated with phosphate dysregulation are detected.
 27. Themethod of claim 26, wherein the morphological changes associated withphosphate dysregulation comprise vacuole-like structures in tumor cells.28. A method of determining whether a subject suffering from cancer hasa tumor sensitive to phosphate dysregulation comprising detecting theexpression of SLC34A2 in a tumor sample obtained from the subject,wherein if SLC34A2 expression is higher in the tumor sample as comparedto expression in normal tissue the tumor is sensitive.
 29. The method ofclaim 28, further comprising detecting PAX8.
 30. A method of determiningwhether a subject suffering from cancer has a tumor sensitive tophosphate dysregulation comprising detecting the amplification in XPR1copy number in a tumor sample obtained from the subject, wherein if XPR1copy number amplification is detected in the tumor sample the tumor issensitive.
 31. The method of claim 30, wherein copy number is detectedby inference from a target sequencing panel at the XPR1 locus onchromosome
 1. 32. The method of any of claims 28 to 31, wherein thecancer is selected from the group consisting of ovarian cancer, uterinecancer, breast cancer, bile duct cancer, liver and lung cancer.
 33. Themethod of any of claims 28 to 32, wherein detecting comprises one ormore of immunohistochemistry (IHC), in situ RNA-seq, quantitative PCR,RNA-seq, CITE-seq, western blot, Fluorescence In Situ Hybridization(FISH), RNA-FISH, mass spectrometry, or FACS.
 34. A method foridentifying an agent capable of inhibiting XPR1:KIDINS220-mediatedphosphate export, comprising: applying a candidate agent to a cancercell or cell population; and detecting modulation of phosphate efflux inthe cell or cell population by the candidate agent, thereby identifyingthe agent.
 35. A method for identifying a cancer sensitive to inhibitionof XPR1:KIDINS220-mediated phosphate export, comprising: applying aninhibitor of XPR1:KIDINS220-mediated phosphate export to a cancer cellor cell population; and detecting the phosphate concentration in thecell or cell population, wherein the cancer is sensitive if thephosphate concentration is increased as compared to a control cell orpopulation not treated with the inhibitor.
 36. The method of claim 35,wherein the inhibitor of XPR1:KIDINS220-mediated phosphate export is oneor more therapeutic agents according to any of claims 4 to
 17. 37. Themethod of claim 35 or 36, wherein the cancer cell or population isobtained or derived from a subject in need thereof.